Detection and quantitation of urine gelsolin

ABSTRACT

The invention relates generally to gelsolin binding agents (e.g., antibodies) which can bind to gelsolin polypeptides. Gelsolin binding agents of the invention are useful, alone or in combination, to detect a gelsolin polypeptide in a test sample. In particular, the gelsolin binding agents are useful in assays of urine samples to diagnose a gelsolin-related medical condition. Kits to detect gelsolin in biological samples are also provided by the present disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application No.PCT/CN2007/002467, filed Aug. 15, 2007, the contents of which areincorporated herein in its entirety.

FIELD OF THE INVENTION

This invention relates generally to methods of detecting urine gelsolin.In particular, the present invention relates to the detection of urinegelsolin-like polypeptides using specific binding agents.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Actin is the most abundant protein in animal cells and constitutes10-20% of the protein of many nucleated cells and 30% of the protein ofmuscle cells. Actin molecules each bind an ATP molecule andself-assemble into long filaments during which the ATP is hydrolyzedinto ADP.

Injury to animal tissues results in the release of actin into theextracellular space, including the bloodstream. Although approximatelyhalf of nonmuscle cell actin is F-actin, (the double-helical, rodlike,filament form of actin which is assembled from G-actin monomers), theionic conditions of extracellular fluids favor actin polymerization, sothat virtually all the actin released into the blood from dying cellswould be expected to polymerize into filaments (Lind, S. E. et al., Am.Rev. Respir. Dis. 138:429-434 (1988)). In purified solutions, in theabsence of filament-shortening proteins, actin filaments can easilyattain lengths of several microns. Were some fraction of actin releasedfrom injured cells to be irreversibly denatured, however, or else boundto one of the intracellular actin-binding proteins discussed below, thisactin would remain monomeric.

There are many proteins which naturally associate with actin (for areview of actin-binding proteins, see Stossel et al., Ann. Rev. CellBiol. 1:353-402 (1985); Pollard et al., Ann. Rev. Biochem. 55:987-1035(1986)). However, two proteins, gelsolin and DBP (vitamin D bindingprotein) are thought to be primarily responsible for bindingextracellular actin. (Janmey et al., Blood 70:529-530 (1987).) Gelsolinis an actin-binding protein that is a key regulator of actin filamentassembly and disassembly. Gelsolin is an 82-kDa protein with sixhomologous subdomains, referred to as S1-S6. Each subdomain is composedof a five-stranded β-sheet, flanked by two α-helices, one positionedperpendicular with respect to the strands and one positioned parallel.The N-terminus (S1-S3) forms an extended β-sheet, as does the C-terminal(S4-S6). (Kiselar et al. PNAS 100:3942-3947 (2003).) The protein ishighly conserved and highly homologous among species. Gelsolin islocated intracellularly (in cytosol and mitochondria) andextracellularly (in blood plasma). Koya et al., J Biol Chem 275(20):15343-15349 (2000).

Gelsolin has several functions in regulating actin polymerization.First, gelsolin is involved in monomeric actin binding. In the presenceof Ca²⁺, gelsolin binds two actin monomers. Gelsolin can also bind actinfilaments by another actin binding site. Second, gelsolin binds twoactin monomers to form a nucleus for actin polymerization and caps thebarbed end of actin filaments. Thus, gelsolin is capable of both servingas a nucleus for actin polymerization and capping the ends of thenascent microfilaments. Finally, gelsolin has actin severing activity.

Because of the large amounts of actin in cells, the release of actinfrom dying cells provides sufficient actin to have a significant affecton the microenvironment, either by increasing the viscosity ofextracellular fluids of plasma and/or by entrapping cells or by other,as yet unidentified toxic effects. Infusion of extracellular free actinis toxic to animal tissues, and especially to renal and cardiopulmonarysystems. (Harper et al., Clin. Res. 36:625 A (1988); Haddad et al., PNAS87: 1381-1385 (1990).) Acute renal failure is a complication of muscleinjury and actin infusions in rats causes transient elevations of theblood urea nitrogen (BUN) and creatinine levels, consistent with renalfailure. Free actin in the plasma may form filaments which may lead tomultiple organ dysfunction syndrome. (Dahl et al., Shock 12(2):102-4(1999).) Moreover, since each extracellular actin molecule in a filamenthas an ADP molecule associated with it, the presence of extracellularactin in the blood may tend to induce or augment platelet aggregation ina manner which may not be advantageous to the host. (Lind et al., Am.Rev. Respir. Dis. 138:429-434 (1988); Scarborough et al., Biochem.Biophy. Res. Commun. 100:1314-1319 (1981).) Consequently, plasmagelsolin has a vital function of scavenging actin released from dead anddying cells and plasma gelsolin levels appear to be an early prognosticmarker in patients experiencing trauma. (Mounzer et al., Am. J. Respir.Crit. Care Med. 160:1673-81 (1999).)

SUMMARY OF THE INVENTION

This invention relates generally to the preparation of gelsolin bindingagents and uses of the same. In particular, the present inventionrelates to a method for determining the presence of, or predispositionto, a disease or condition associated with altered levels of gelsolin ina mammalian subject, the method comprising the steps of: (a) contactinga urine sample from the mammalian subject under immunologically-reactiveconditions with one or more antibodies or antibody-related polypeptideshaving the same antigen-binding specificity as antibodies produced by adeposited cell line selected from the group consisting of: CGMCCAccession Nos: 2114, 2116, 2247, and 2248; and (b) detecting the bindingof the one or more antibodies or antibody-related polypeptides to thegelsolin-like polypeptide to determine the level of gelsolin-likepolypeptide in the sample, wherein a difference between the level ofgelsolin-like polypeptide in the sample and a reference level indicatesa presence of, or predisposition to, a disease or condition associatedwith altered levels of gelsolin in the mammalian subject.

In one embodiment, the sample is contacted with the antibodies orantibody-related polypeptides in an ELISA. The step of contact maycomprise binding a first antibody to a substrate and contacting thesample and a second antibody to the substrate, wherein the secondantibody comprises a detectable label. In one embodiment, the firstantibody binds to the same antigenic determinant as an antibody producedby a hybridoma cell line CGMCC Accession No: 2247 and the secondantibody binds to the same antigenic determinant as an antibody producedby a hybridoma cell line selected from the group consisting of CGMCCAccession No. 2116.

The instant methods may be useful to diagnose a disease or conditionassociated with altered levels of gelsolin. The disease or conditionassociated with altered levels of gelsolin may be selected from thegroup consisting of: kidney failure, septic shock, multiple organdysfunction syndrome, rheumatoid arthritis, stroke, heart infarction,cancer, systemic autoimmune disease, chronic hepatitis, side-effects ofchemotherapy, and side-effects of radiation therapy.

In some embodiments, the gelsolin-like polypeptide being detected inurine gelsolin is a C-terminal fragment of gelsolin. For instance, anincrease in the level of gelsolin-like polypeptide in the samplecompared to the reference level indicates a presence of, orpredisposition to, a disease or condition associated with altered levelsof gelsolin in a mammalian subject.

In one embodiment, the reference level is the amount of thegelsolin-like polypeptide in a control population of subjects that donot have a disease or condition associated with altered levels ofgelsolin. In another embodiment, the reference level is the amount ofthe gelsolin-like polypeptide in a subject that does not have a diseaseor condition associated with altered levels of gelsolin. In yet anotherembodiment, the reference level is the amount of the gelsolin-likepolypeptide in the subject at an earlier time.

In one embodiment, the methods further comprise quantifying the amountof the gelsolin-like polypeptide in the sample from the subject. In oneembodiment, the methods further comprise correlating the amount ofgelsolin-like polypeptide in the sample from the mammalian subject to alikelihood of survival of the subject.

In another aspect, the invention provides a method for monitoring kidneyfailure in a mammalian subject, the method comprising the steps of: (a)determining the level of gelsolin-like polypeptide in the urine of amammalian subject; (b) comparing the level of gelsolin-like polypeptidein the mammalian subject to a reference level, wherein the referencelevel comprises a control subject not having kidney failure, and whereinan increase in the level of gelsolin-like polypeptide in the subjectcompared to the reference level indicates that the mammalian subject haskidney failure.

In another aspect, the invention provides a method for selecting aprophylactic or therapeutic treatment for a mammalian subject,comprising the steps of: (a) determining the level of gelsolin-likepolypeptide in the mammalian subject; (b) assigning the subject to asubject class based on the level of gelsolin polypeptide of the subject;and (c) selecting a prophylactic or therapeutic treatment based on thesubject class.

In another aspect, the invention provides a method of selecting amammalian subject for inclusion in a clinical trial for determining theefficacy of a compound to prevent or treat a medical conditionassociated with altered levels of gelsolin in a mammalian subject,comprising the steps of: (a) measuring the level of gelsolin-likepolypeptide in a sample from the mammalian subject; (b) comparing thelevel of gelsolin in the subject to a reference level, wherein thereference level comprises a control population not having a disease orcondition associated with altered levels of gelsolin; and (c) selectingto include the mammalian subject in the clinical trial, wherein asimilarity in the level of the gelsolin-like polypeptide in the samefrom the mammalian subject is similar to the level in the referencestandard.

In another aspect, the invention provides an antibody or antigen-bindingfragment thereof having the same antigen-binding specificity ofantibodies produced by a deposited cell line selected from the groupconsisting of: CGMCC Accession Nos: 2247 and 2248. In one embodiment,the invention provides an isolated nucleic acids encoding the antibodyor an antigen-binding fragment thereof. In one embodiment, the isolatednucleic acids encoding the antibody or an antigen-binding fragmentthereof are contained in a vector. The vector may also comprise apromoter operably-linked to the nucleic acid molecule to directexpression of the nucleic acid encoding the antibody or anantigen-binding fragment thereof are contained in a vector. In oneaspect, the invention provides host cells comprising the vectorsencoding the antibody or an antigen-binding fragment thereof.

In one aspect, the present invention provides a continuous cell linewhich produces a monoclonal antibody, wherein said monoclonal antibodybinds to the same antigenic determinant as an antibody produced by ahybridoma cell line selected from the group consisting of: CGMCCAccession Nos: 2247 and 2248, wherein the cell line is produced by theprocess of fusing a lymphocyte derived from a mouse immunized withcarcinoma cells or an immunogenic determinant thereof and a mousemyeloma cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a SDS-PAGE analysis of human urine gelsolinimmuno-precipitated with anti-gelsolin antibodies. The samples in eachlane were as follows: lanes 1-3: healthy control patients; lanes 4-8:severe stroke (ICU) patients. The top panel depicts samplesimmuno-precipitated with N-terminal specific anti-gelsolin antibodyGN3E9. The bottom panel depicts samples immuno-precipitated withC-terminal specific anti-gelsolin antibody GC1C10.

FIG. 2 is a western blot of SDS-PAGE of immuno-precipitated urineproteins in healthy control subjects (lanes 1-3) or ICU patients (lanes4-8). FIG. 2A depicts samples immuno-precipitated with N-terminalspecific anti-gelsolin antibody GN3E9 and detected using N-terminalspecific antibody GN3E9. FIG. 2B depicts samples immuno-precipitatedwith C-terminal specific anti-gelsolin antibody GC1C10 and detectedusing C-terminal specific antibody GC1C10.

FIG. 3 is a western blot of SDS-PAGE of immuno-precipitated urineproteins in healthy control subjects (lanes 1-3) or ICU patients (lanes4-8). Samples were immuno-precipitated anti-gelsolin antibody GC5D1 anddetected using C-terminal specific antibody GF2D6.

FIG. 4 is a graph of optical density versus gelsolin fragmentconcentration, which shows the quantitative determination of full-length(FL), N-terminal specific (NT), or C-terminal (CT) gelsolin fragments byan ELISA assay using GC5D1 was the capture antibody and C-terminalspecific antibody GF2D6 as the detection antibody.

FIG. 5A and FIG. 5B are graphs of urine gelsolin concentration incontrol subjects and patients having various forms of trauma measuredusing a gelsolin ELISA assay of the invention. The C-terminal specificantibody GC5D1 was the capture antibody and C-terminal specific antibodyGF2D6 as the detection antibody.

FIG. 6 is a graph showing the correlation between serum gelsolin levelsand urine gelsolin-like polypeptides in patient-matched samples measuredusing a gelsolin ELISA assay of the invention.

DETAILED DESCRIPTION

General.

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

The invention generally provides gelsolin binding agents (e.g.,antibodies) which can bind to gelsolin polypeptides. Gelsolin bindingagents of the invention are useful, alone or in combination, to detect agelsolin polypeptide (a.k.a., the target polypeptide) in a test sampleas well as in methods to purify gelsolin proteins from a biologicalsample. Gelsolin binding agents are useful to diagnose agelsolin-related medical condition in subjects in need thereof. An aminoacid sequence of a human gelsolin polypeptide (SEQ ID NO.: 1) is shownin Table 1.

TABLE 1 Human Gelsolin Polypeptide Sequence (SEQ ID NO.: 1)MAPHRPAPALLCALSLALCALSLPVRAATASRGASQAGAPQGRVPEARPNSMVVEHPEFLKAGKEPGLQIWRVEKFDLVPVPTNLYGDFFTGDAYVILKTVQLRNGNLQYDLHYWLGNECSQDESGAAAIFTVQLDDYLNGRAVQHREVQGFESATFLGYFKSGLKYKKGGVASGFKHVVPNEVVVQRLFQVKGRRVVRATEVPVSWESFNNGDCFILDLGNNIHQWCGSNSNRYERLKATQVSKGIRDNERSGRARVHVSEEGTEPEAMLQVLGPKPALPAGTEDTAKEDAANRKLAKLYKVSNGAGTMSVSLVADENPFAQGALKSEDCFILDHGKDGKIFVWKGKQANTEERKAALKTASDFITKMDYPKQTQVSVLPEGGETPLFKQFFKNWRDPDQTDGLGLSYLSSHIANVERVPFDAATLHTSTAMAAQHGMDDDGTGQKQIWRIEGSNKVPVDPATYGQFYGGDSYIILYNYRHGGRQGQIIYNWQGAQSTQDEVAASAILTAQLDEELGGTPVQSRVVQGKEPAHLMSLFGGKPMIIYKGGTSREGGQTAPASTRLFQVRANSAGATRAVEVLPKAGALNSNDAFVLKTPSAAYLWVGTGASEAEKTGAQELLRVLRAQPVQVAEGSEPDGFWEALGGKAAYRTSPRLKDKKMDAHPPRLFACSNKIGRFVIEEVPGELMQEDLATDDVMLLDTWDQVFVWVGKDSQEEEKTEALTSAKRYIETDPANRDRRTPITVVKQGFEPPSFVGWFLGWDDDYWSVDPLDRAMAELAA

In some embodiments, the gelsolin binding agents (e.g., anti-gelsolin oranti-gelsolin-like antibodies) of the present invention detect theactive, or unbound, form of gelsolin. While not wishing to be limited bytheory, free and complexed (to actin) gelsolin molecules differ in theirfunctional properties. Although free gelsolin can sever actin filaments,actin-gelsolin in complexes cannot. Gelsolin's severing activity isactivated by micromolar Ca²⁺ and has been shown to be inhibited byphosphatidyl inositol bisphosphate (PIP₂) and phosphatidyl inositolmonophosphate (PIP). Since extracellular Ca²⁺ concentrations are atmillimolar levels and extracellular fluids do not normally contain PIPor PIP₂ in a form that inhibits gelsolin, plasma gelsolin isconstitutively active in extracellular fluids.

The various aspects of the present invention further relate todiagnostic methods and kits that use the gelsolin binding agents of theinvention to identify individuals predisposed to a medical condition orto classify individuals with regard to drug responsiveness, sideeffects, or optimal drug dose. In other aspects, the invention providesmethods for purifying gelsolin or gelsolin-like polypeptides from abiological sample, including, for example, native human gelsolin fromplasma. Accordingly, various particular embodiments that illustratethese aspects follow.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonuchotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, New York (1987)); and Meth. Enzymol., Vols. 154 and 155, Wu& Grossman, and Wu, Eds., respectively. Methods to detect and measurelevels of polypeptide gene expression products (i.e., gene translationlevel) are well-known in the art and include the use polypeptidedetection methods such as antibody detection and quantificationtechniques. (See also, Strachan & Read, Human Molecular Genetics, SecondEdition. (John Wiley and Sons, Inc., New York (1999).)

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. As used inthis specification and the appended claims, the singular forms “a,” “an”and “the” include plural referents unless the content clearly dictatesotherwise. For example, reference to “a cell” includes a combination oftwo or more cells, and the like. Generally, the nomenclature used hereinand the laboratory procedures in cell culture, molecular genetics,organic chemistry, analytical chemistry and nucleic acid chemistry andhybridization described below are those well-known and commonly employedin the art. All references cited herein are incorporated herein byreference in their entireties and for all purposes to the same extent asif each individual publication, patent, or patent application wasspecifically and individually incorporated by reference in its entiretyfor all purposes.

Definitions.

The definitions of certain terms as used in this specification areprovided below. Definitions of other terms may be found in theIllustrated Dictionary of Immunology, 2nd Edition (Cruse, J. M. andLewis, R. E., Eds., Boca Raton, Fla.: CRC Press, 1995). Unless indicatedotherwise, the term “gelsolin” when used herein refer to the humanprotein and gene.

As used herein, the “administration” of an agent or drug to a subject orsubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),rectally, or topically. Administration includes self-administration andthe administration by another. It is also to be appreciated that thevarious modes of treatment or prevention of medical conditions asdescribed are intended to mean “substantial,” which includes total butalso less than total treatment or prevention, and wherein somebiologically or medically relevant result is achieved.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “antibody” means a polypeptide comprising aframework region from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes an antigen, e.g., a gelsolinpolypeptide. Use of the term antibody is meant to include wholeantibodies, including single-chain whole antibodies, and antigen-bindingfragments thereof. The term “antibody” includes bispecific antibodiesand multispecific antibodies so long as they exhibit the desiredbiological activity or function.

As used herein, the term “antibody-related polypeptide” meansantigen-binding antibody fragments, including single-chain antibodies,that can comprise the variable region(s) alone, or in combination, withall or part of the following polypeptide elements hinge region, CH₁,CH₂, and CH₃ domains of an antibody molecule. Also included in theinvention are any combinations of variable region(s) and hinge region,CH₁, CH₂, and CH₃ domains. Antibody-related molecules useful as bindingagents of the invention include, e.g., but are not limited to, Fab, Fab′and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. Examples include: (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH₁ domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the V_(H) and CH₁ domains; (iv) a Fv fragment consistingof the V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAbfragment (Ward et al., Nature 341:544-546, (1989)), which consists of aV_(H) domain; and (vi) an isolated complementarity determining region(CDR). As such “antibody fragments” can comprise a portion of a fulllength antibody, generally the antigen binding or variable regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Single-chain antibody molecules may comprise a polymer with a number ofindividual molecules, for example, dimer, trimer or other polymers.

As used herein, the term “CDR-grafted antibody” means an antibody inwhich at least one CDR of an “acceptor” antibody is replaced by a CDR“graft” from a “donor” antibody possessing a desirable antigenspecificity.

As used herein, the term “chimeric antibody” means an antibody in whichthe Fc constant region of a monoclonal antibody from one species (e.g.,a mouse Fc constant region) is replaced, using recombinant DNAtechniques, with an Fc constant region from an antibody of anotherspecies (e.g., a human Fc constant region). See generally, Robinson etal., PCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European PatentApplication 125,023; Better et al., Science 240:1041-1043 (1988); Liu etal., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu et al., J.Immunol 139:3521-3526 (1987); Sun et al., Proc. Natl. Acad. Sci. USA84:214-218 (1987; Nishimura et al., Cancer Res 47:999-1005 (1987); Woodet al., Nature 314:446-449 (1985); and Shaw et al., J. Natl. CancerInst. 80:1553-1559 (1988).

As used herein, the term “clinical response” means any or all of thefollowing: a quantitative measure of the response, no response, andadverse response (i.e., side effects).

As used here, in the term “clinical trial” means any research studydesigned to collect clinical data on responses to a particulartreatment, and includes, but is not limited to phase I, phase II, andphase III clinical trials. Standard methods are used to define thepatient population and to enroll subjects.

As used herein, the term “consensus FR” means a framework (FR) antibodyregion in a consensus immunoglobulin sequence. The FR regions of anantibody do not contact the antigen.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) in the same polypeptide chain (V_(H) V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen binding sites. Diabodies aredescribed more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, the term “effector cell” means an immune cell which isinvolved in the effector phase of an immune response, as opposed to thecognitive and activation phases of an immune response. Exemplary immunecells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes(e.g., B cells and T cells including cytolytic T cells (CTLs)), killercells, natural killer cells, macrophages, monocytes, eosinophils,neutrophils, polymorphonuclear cells, granulocytes, mast cells, andbasophils. Effector cells express specific Fc receptors and carry outspecific immune functions. An effector cell can induceantibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophilcapable of inducing ADCC. For example, monocytes, macrophages,neutrophils, eosinophils, and lymphocytes which express FcαR areinvolved in specific killing of target cells and presenting antigens toother components of the immune system, or binding to cells that presentantigens. An effector cell can also phagocytose a target antigen, targetcell, metastatic cancer cell, or microorganism.

As used herein, the term “epitope” means a protein determinant capableof specific binding to an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. In one embodiment, an “epitope” of gelsolin is aregion in the gelsolin protein to which the gelsolin binding agent ofthe invention binds. In select embodiments of the invention, thisepitope is in the domain spanning amino acid residues from about 321 toabout 330, from about 636 to about 645, or from about 661 to 670 of SEQID NO.: 1.

To screen for gelsolin binding agents which bind to an epitope, aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. This assay can be used to determine if agelsolin binding agent binds the same site or epitope as a gelsolinantibody of the invention. Alternatively, or additionally, epitopemapping can be performed by methods known in the art. For example, theantibody sequence can be mutagenized such as by alanine scanning, toidentify contact residues. In a different method, peptides correspondingto different regions of gelsolin can be used in competition assays withthe test antibodies or with a test antibody and an antibody with acharacterized or known epitope.

As used herein, the term “effective amount” or “pharmaceuticallyeffective amount” or “therapeutically effective amount” of acomposition, is a quantity sufficient to achieve a desired therapeuticand/or prophylactic effect, e.g., an amount which results in theprevention of, or a decrease in, the symptoms associated with a diseasethat is being treated, e.g., the diseases or medical conditionsassociated with target polypeptide (e.g., gelsolin or gelsolin-likepolypeptides). The amount of a composition of the invention administeredto the subject will depend on the type and severity of the disease andon the characteristics of the individual, such as general health, age,sex, body weight and tolerance to drugs. It will also depend on thedegree, severity and type of disease. The skilled artisan will be ableto determine appropriate dosages depending on these and other factors.The compositions of the present invention can also be administered incombination with one or more additional therapeutic compounds. In themethods of the present invention, gelsolin may be administered to asubject having decreased gelsolin levels caused by a disease ortraumatic condition, thereby increasing the level of plasma gelsolin inthe subject. For example, a “therapeutically effective amount” ofgelsolin is meant levels in which the toxic effects of freeextracellular actin are, at a minimum, ameliorated.

As used herein, “expression” includes but is not limited to one or moreof the following: transcription of the gene into precursor mRNA;splicing and other processing of the precursor mRNA to produce maturemRNA; mRNA stability; translation of the mature mRNA into protein(including codon usage and tRNA availability); and glycosylation and/orother modifications of the translation product, if required for properexpression and function.

As used herein, the term “gelsolin” refers to a multifunctional actinbinding protein. In mammals, gelsolin comprises two isoforms:cytoplasmic and extracellular variants. Human plasma gelsolin differsfrom cellular gelsolin only by the addition of about 25 amino acids tothe N-terminus of the molecule and both gelsolins are the product of asingle gene. Plasma gelsolin has three actin-binding sites and bindswith high affinity to either G-actin or F-actin. “Gelsolin” also refersto recombinant forms of the mammalian polypeptide.

As used herein, a “gelsolin-like polypeptide” means a polypeptide thatis different from gelsolin polypeptide but which isimmunologically-reactive with a gelsolin binding agent of the invention.A gelsolin-like polypeptide may be derived from the same organism or adifferent organism as a gelsolin polypeptide. A gelsolin-likepolypeptide may be encoded by the same gene or a different gene as agelsolin polypeptide. A gelsolin-like polypeptide may be animmunoreactive fragment (e.g., a C-terminal fragment) of a gelsolinpolypeptide.

As used herein, the term “gene” means a segment of DNA that contains allthe information for the regulated biosynthesis of an RNA product,including promoters, exons, introns, and other untranslated regions thatcontrol expression.

As used herein, the term “human sequence antibody” includes antibodieshaving variable and constant regions (if present) derived from humangermline immunoglobulin sequences. The human sequence antibodies of theinvention can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo). Suchantibodies can be generated in non-human transgenic animals, e.g., asdescribed in PCT Publication Nos. WO 01/14424 and WO 00/37504. However,the term “human sequence antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (e.g., humanized antibodies).

As used herein, the term “humanized” forms of non-human (e.g., murine)antibodies are chimeric antibodies which contain minimal sequencederived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance such asbinding affinity. Generally, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence although theFR regions may include one or more amino acid substitutions that improvebinding affinity. The number of these amino acid substitutions in the FRare typically no more than 6 in the H chain, and in the L chain, no morethan 3. The humanized antibody optionally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Amino acid sequence modification(s)” of the gelsolin antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of a gelsolin antibody areprepared by introducing appropriate nucleotide changes into the antibodynucleic acid, or by peptide synthesis. Such modifications include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the gelsolin antibody. Anycombination of deletion, insertion, and substitution is made to obtainthe antibody of interest, as long as the obtained antibody possesses thedesired properties. The modification also includes the change of thepattern of glycosylation of the protein. A useful method foridentification of preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells in Science,244:1081-1085 (1989). The mutated antibody is then screened for thedesired activity. The invention includes antibody variants with one ormore amino acid addition, deletion and/or substitution of the amino acidsequence defined by hybridomas GC1C10, CN3E9, GF2D6, GC5D1, GC4A10having CGMCC Accession Numbers 2114, 2115, 2116, 2247 and 2248,respectively, provided that the antibody variant possesses the desiredproperties.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (e.g., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 52A-55(H2) and 96-101 (H3) in the V_(H) (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

As used herein, the terms “identical” or percent “identity”, when usedin the context of two or more nucleic acids or polypeptide sequences,refers to two or more sequences or subsequences that are the same orhave a specified percentage of amino acid residues or nucleotides thatare the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region (e.g., nucleotide sequence encoding anantibody described herein or amino acid sequence of an antibodydescribed herein), when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site). Such sequences are then said to be “substantiallyidentical.” This term also refers to, or can be applied to, thecomplement of a test sequence. The term also includes sequences thathave deletions and/or additions, as well as those that havesubstitutions. The algorithms can account for gaps and the like.Typically, identity exists over a region that is at least about 25 aminoacids or nucleotides in length, or over a region that is at least about50-100 amino acids or nucleotides in length.

An “isolated” or “purified” polypeptide or biologically-active portionthereof is substantially free of cellular material or othercontaminating polypeptides from the cell or tissue source from which thegelsolin binding agent is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. For example,an isolated gelsolin binding agent which is an anti-gelsolin oranti-gelsolin-like antibody would be free of materials that wouldinterfere with diagnostic or therapeutic uses of the agent. Suchinterfering materials may include enzymes, hormones and otherproteinaceous and nonproteinaceous solutes. Alternatively, an isolatedgelsolin or gelsolin-like polypeptide, which is immunoractive with agelsolin binding agent of the invention, would be substantially free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe polypeptide.

As used herein, the term “intact antibody” means an antibody that has atleast two heavy (H) chain polypeptides and two light (L) chainpolypeptides interconnected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor V_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH₁, CH₂ and CH₃. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyl-terminus in the following order: FR₁, CDR₁,FR₂, CDR₂, FR₃, CDR₃, FR₄. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

As used herein, the terms “immunologically cross-reactive” and“immunologically-reactive” are used interchangeably to mean an antigenwhich is specifically reactive with an antibody which was generatedusing the same (“immunologically-reactive”) or different(“immunologically cross-reactive”) antigen. Generally, the antigen isgelsolin or gelsolin-like polypeptide, a variant or subsequence thereof.

As used herein, the term “immunologically-reactive conditions” meansconditions which allow an antibody, generated to a particular epitope ofan antigen, to bind to that epitope to a detectably greater degree thanthe antibody binds to substantially all other epitopes, generally atleast two times above background binding, preferably at least five timesabove background. Immunologically-reactive conditions are dependent uponthe format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See, Harlow & Lane, Antibodies, ALaboratory Manual (Cold Spring Harbor Publications, New York (1988)) fora description of immunoassay formats and conditions.

As used herein, the term “medical condition” includes, but is notlimited to, any condition or disease manifested as one or more physicaland/or psychological symptoms for which treatment and/or prevention isdesirable, and includes previously and newly identified diseases andother disorders. For example, a medical condition may be hepatitis, SLE,cancer, kidney failure, septic shock, stroke, cancer, heart infarction,and side effects of chemotherapy and radiation therapy.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. For example, a monoclonal antibody can be an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.A monoclonal antibody composition displays a single binding specificityand affinity for a particular epitope. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. Furthermore,in contrast to conventional (polyclonal) antibody preparations whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including, e.g., but not limited to, hybridoma,recombinant, and phage display technologies. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

As used herein, the term “pharmaceutically-acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal compounds, isotonic and absorption delayingcompounds, and the like, compatible with pharmaceutical administration.

As used herein, the term “polyclonal antibody” means a preparation ofantibodies derived from at least two (2) different antibody-producingcell lines. The use of this term includes preparations of at least two(2) antibodies that contain antibodies that specifically bind todifferent epitopes or regions of an antigen.

As used herein, the term “polynucleotide” or “nucleic acid” means anyRNA or DNA, which may be unmodified or modified RNA or DNA.Polynucleotides include, without limitation, single- and double-strandedDNA, DNA that is a mixture of single- and double-stranded regions,single- and double-stranded RNA, RNA that is mixture of single- anddouble-stranded regions, and hybrid molecules comprising DNA and RNAthat may be single-stranded or, more typically, double-stranded or amixture of single- and double-stranded regions. In addition,polynucleotide refers to triple-stranded regions comprising RNA or DNAor both RNA and DNA. The term polynucleotide also includes DNAs or RNAscontaining one or more modified bases and DNAs or RNAs with backbonesmodified for stability or for other reasons. In a particular embodiment,the polynucleotide contains polynucleotide sequences from a gelsolingene.

As used herein, the terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature. In aparticular embodiment, the polypeptide contains polypeptide sequencesfrom a gelsolin protein.

As used herein, the term “population” may be any group of at least twoindividuals. A population may include, e.g., but is not limited to, areference population, a population group, a family population, aclinical population, and a same sex population.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the material is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all.

As used herein, the term “reference level” refers to an amount orconcentration of biomarker (e.g., the level of gelsolin or gelsolin-likepolypeptide) which may be of interest for comparative purposes. In oneembodiment, a reference level may be the level of at least one biomarkerexpressed as an average of the level of at least one biomarker takenfrom a control population of healthy subjects. In another embodiment,the reference level may be the level of at least one biomarker in thesame subject at an earlier time, i.e., before the present assay. In evenanother embodiment, the reference level may be the level of at least onebiomarker in the subject prior to receiving a treatment regime.

As used herein, the term “reference standard” is the pattern ofexpression of one or more genes or proteins observed in either areference standard population or a single subject prior toadministration of a compound. A “reference standard population” means apopulation characterized by one or more biological characteristics,e.g., drug responsiveness, genotype, haplotype, phenotype, etc.

As used herein, the phrase “salvage receptor binding epitope” refers toan epitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃,or IgG₄) that is responsible for increasing the in vivo serum half-lifeof the IgG molecule. To increase the serum half life of the antibody,one may incorporate a salvage receptor binding epitope into the antibody(especially an antibody fragment) as described in U.S. Pat. No.5,739,277, for example.

As used herein, the term “sample” means sample material derived from orcontacted by living cells. The term “sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Biological samplesof the invention include, e.g., but are not limited to, whole blood,plasma, semen, saliva, tears, urine, fecal material, sweat, buccal,skin, cerebrospinal fluid, and hair. Biological samples can also beobtained from biopsies of internal organs or from cancers. Biologicalsamples can be obtained from subjects for diagnosis or research or canbe obtained from undiseased individuals, as controls or for basicresearch.

As used herein, the terms “single chain antibodies” or “single chain Fv(scFv)” refer to an antibody fusion molecule of the two domains of theFv fragment, V_(L) and V_(H). Although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv). See, e.g., Bird et al., Science 242:423-426 (1988); and Hustonet al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Such singlechain antibodies are included by reference to the term “antibody”fragments, and can be prepared by recombinant techniques or enzymatic orchemical cleavage of intact antibodies.

As used herein, the term “specific binding” means the contact between agelsolin binding agent and an antigen with a binding affinity of atleast 10⁻⁶ M. Preferred binding agents bind with affinities of at leastabout 10⁻⁷ M, and preferably 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or10⁻¹² M.

As used herein, the term “subject” means that the subject is a mammal,such as a human, but can also be an animal, e.g., domestic animals(e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs,horses and the like) and laboratory animals (e.g., monkey, rats, mice,rabbits, guinea pigs and the like).

As used herein, the term “substitution” is one of mutations that isgenerally used in the art. Those substitution variants have at least oneamino acid residue in the gelsolin binding agent replaced by a differentresidue. The sites of greatest interest for substitutional mutagenesisinclude the hypervariable regions, but FR alterations are alsocontemplated. “Conservative substitutions” are shown in the table belowunder the heading of “preferred substitutions.” If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated “exemplary substitutions” in Table 2, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 2 Amino Acid Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile Val Arg (R) lys; gln;asn Lys Asn (N) gln; his; asp, lys; arg Gln Asp (D) glu; asn Glu Cys (C)ser; ala Ser Gln (Q) asn; glu Asn Glu (E) asp; gln Asp Gly (G) Ala AlaHis (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; Leu phe;norleucine Leu (L) norleucine; ile; val; Ile met; ala; phe Lys (K) arg;gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu; val; ile; ala; tyrTyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) tyr; phe TyrTyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; Leu ala;norleucine

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Specifically, severalhypervariable region sites (e.g., 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibody variantsthus generated are displayed in a monovalent fashion from filamentousphage particles as fusions to the gene III product of M13 packagedwithin each particle. The phage-displayed variants are then screened fortheir biological activity (e.g., binding affinity) as herein disclosed.In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding gelsolin. Alternatively, or additionally, it may be beneficialto analyze a crystal structure of the antigen-antibody complex toidentify contact points between the antibody and gelsolin. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with similar or superior properties in one or morerelevant assays may be selected for further development. The inventionincludes antibody variants with one or more amino acid substitution(s),especially conservative substitutions, to the hypervariable domains ofthe immunoglobulin heavy or light chain defined by hybridomas GC1C10,GN3E9, GF2D6, GC5D1, and GC4A10 having CGMCC Accession Numbers 2114,2115, 2116, 2247, and 2248, respectively, provided that the antibodyvariant possesses the desired properties.

As used herein, a “test sample” means a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., serum or urine), cell or tissue sample, sample from cultureor growth media, or isolated nucleic acid or polypeptide derivedtherefrom.

As used herein, the term “therapeutic agent” is intended to mean acompound that, when present in an effective amount, produces a desiredtherapeutic effect on a subject in need thereof.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for a disorder characterized by decreased gelsolin levels if,after receiving a therapeutic amount of native or recombinant gelsolinaccording to the methods of the present invention, the subject showsobservable and/or measurable reduction in or absence of one or moresigns and symptoms of a particular disease or condition associated withaltered serum gelsolin levels.

As used herein, the term “variable” refers to the fact that certainsegments of the variable domains differ extensively in sequence amongantibodies. The V domain mediates antigen binding and define specificityof a particular antibody for its particular antigen. However, thevariability is not evenly distributed across the amino acid span of thevariable domains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies. (See Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991).) The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

I. Compositions of the Invention

A. Gelsolin Binding Agents.

In one aspect, the present invention provides gelsolin binding agentcompositions, a.k.a., the binding agent. In one embodiment, the bindingagent of the invention is an intact antibody directed to gelsolin, agelsolin-like polypeptide, homolog, fragment, or derivative thereof. Thebinding agents of interest may be ones which bind specifically to free,full-length, active gelsolin, but do not “substantively” (or“substantially”) bind gelsolin which are bound to actin. In suchembodiments, the extent of binding of the binding agent of the inventionto these proteins will be less than about 10%, preferably, or less thanabout 5%, or less than about 1%, as determined by fluorescence activatedcell sorting (FACS) analysis, ELISA, Western blot, or radioimmunoassay.Alternatively, the binding agents (or combination thereof) may bind afragment of gelsolin (e.g., a C-terminal fragment), but do notsubstantially bind to full-length gelsolin.

Prior efforts to generate monoclonal antibodies with defined epitopes(in particular, eptiopes associated with functional gelsolin) have beenlargely unsuccessful. Gelsolin is a highly conserved protein and highlyhomologous among species. Gelsolin is also abundant in plasma, whichrequires that it be well-tolerated by the immune system. Furthermore,due to the fact that gelsolin is a major actin binding protein, theexposed epitopes are limited by the complexing of gelsolin with actinand other plasma proteins. Although some monoclonal antibodies togelsolin have been produced (see Hiyoshi et al., Biochem Mol Biol Int32:755-62 (1994)), there is no immunoassay for quantitative measurementof plasma gelsolin.

The present inventors have discovered a strategy to design gelsolinbinding agents using various forms of human gelsolin proteins for bothimmunization and screening, including native gelsolin, recombinantfull-length gelsolin, and N- and C-terminal gelsolin fragments.Moreover, the inventors' strategy is designed to break the immunetolerance to common epitopes of human gelsolin using modulators of theimmune response. The result of this strategy are gelsolin binding agentswith defined epitopes that allow for rapid, accurate, and quantitativeassays for plasma gelsolin and/or urine gelsolin-like polypeptides(i.e., gelsolin fragments), which can be used in a clinical setting.

Binding agents of the present invention can be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the bindingagent, e.g., a region of the gelsolin polypeptide that is located on thesurface of the polypeptide (e.g., a hydrophilic region). In oneembodiment, the invention provides gelsolin binding agents, e.g.,antibodies or antibody-related polypeptides directed to a gelsolinpolypeptide (a.k.a., a target polypeptide) comprising one or more aminoacid sequences selected from the group consisting of: FAQGALKSED (SEQ IDNO.: 2), SEPDGFWEAL (SEQ ID NO.: 3), and ACSNKIGRFV (SEQ ID NO.: 4).

In select embodiments, the invention provides the gelsolin bindingagents summarized in Table 3.

TABLE 3 Select Gelsolin Binding Agents Binding Agent Type DescriptionGN3E9 Murine   Murine monoclonal antibody  Monoclonaldirected to an epitope com-  Antibody prising a polypeptide   sequence of FAQGALKSED (SEQ ID NO.: 2). GC1C10 Murine Murine monoclonal antibody  Monoclonal directed to an epitope com-  Antibody prising a polypeptide   sequence of SEPDGFWEAL (SEQ ID NO.: 3). GF2D6 Murine   Murine monoclonal antibody  Monoclonaldirected to a epitope with  Antibody a polypeptide sequence of ACSNKIGRFV (SEQ ID NO.: 4). GC5D1 Murine  Murine monoclonal antibody  Monoclonal directed to a epitope with  Antibodya polypeptide sequence of  GASEAEKTGA (SEQ ID NO.: 5). GC4A10 Murine  Murine monoclonal antibody  Monoclonal directed to a epitope with Antibody a polypeptide sequence of  GASEAEKTGA (SEQ ID NO.: 5).

Deposits of biological materials associated with the gelsolin bindingagents summarized in Table 3 (above) were made with the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, P.O. Box 2714, Beijing 100080, ThePeople's Republic of China as detailed in Table 4 below.

TABLE 4 Biological Deposits Name of Accession Deposit Materials DateNumber GN3E9 Mouse-mouse Jul. 20, 2007 2115 hybridoma GC1C10 Mouse-mouseJul. 20, 2007 2114 hybridoma GF2D6 Mouse-mouse Jul. 20, 2007 2116hybridoma GC5D1 Mouse-mouse Nov. 6, 2007 2247 hybridoma GC4A10Mouse-mouse Nov. 6, 2007 2248 hybridoma

In another embodiment, the present invention affords a method ofelucidating other epitopes of gelsolin, which can be used for generationof an antibody having the desired characteristics of binding to activegelsolin, but not gelsolin bound to actin. The binding agents directedagainst the epitope may have a differing variable or CDR region butshould have the binding and functional characteristics of the antibodiesof the present invention. As a means for targeting antibody production,hydropathy plots showing regions of hydrophilicity and hydrophobicitycan be generated by any method well known in the art, including, e.g.,the Kyte Doolittle or the Hopp Woods methods, either with or withoutFourier transformation. (See, e.g., Hopp and Woods, Proc. Nat. Acad.Sci. USA 78: 3824-3828 (1981); Kyte and Doolittle, J. Mol. Biol.157:105-142 (1982).) The epitope(s) or polypeptide portion(s) can bespecified as described herein, e.g., by N-terminal and C-terminalpositions, by size in contiguous amino acid residues. The presentinvention includes binding agents that specifically bind polypeptides ofthe present invention, and allows for the exclusion of the same. Thepresent invention includes binding agents that specifically bindepitopes which are conformational epitopes or nonconformationalepitopes. As noted above, conformational epitopes or nonconformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents.

Binding agents of the present invention can also be described orspecified in terms of their cross-reactivity. Binding agents that do notbind any other analog, ortholog, or homolog of the target polypeptide ofthe present invention are included. Binding agents that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. Further includedin the present invention are binding agents which only bind polypeptidesencoded by polynucleotides which hybridize to a polynucleotide of thepresent invention under stringent hybridization conditions (as describedherein).

Binding agents of the present invention can also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or K_(d) less than5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M. In one embodiment,the invention provides gelsolin binding agents that at least bind humangelsolin with a K_(d) value of no higher than 1×10⁻⁸, preferably a K_(d)value no higher than about 1×10⁻⁹.

Gelsolin binding agents within the scope of the present inventioninclude, e.g., but are not limited to, monoclonal, polyclonal, chimeric,humanized, diabody, and human monoclonal and human polyclonal antibodieswhich specifically bind the target polypeptide, a homolog, derivative ora fragment thereof. The antibodies useful as binding agents of thepresent invention include, e.g., but are not limited to, IgG (includingIgG₁, IgG₂, IgG₃, and IgG₄), IgA (including IgA₁ and IgA₂), IgD, IgE, orIgM, and IgY.

In another embodiment, the binding agent of the invention is anantibody-related polypeptide directed to gelsolin polypeptide, homologor derivative thereof. Typically, the antigen-binding region of abinding agent, e.g., the anti-gelsolin binding region, will be mostcritical in specificity and affinity of binding of the binding agent ofthe invention. In some embodiments, the gelsolin binding agent is ananti-gelsolin polypeptide antibody, such as an anti-gelsolin polypeptidemonoclonal antibody, an anti-gelsolin polypeptide chimeric antibody, andan anti-gelsolin polypeptide humanized antibody which have been modifiedby, e.g., deleting, adding, or substituting portions of the antibody.

In one embodiment, selection of antibodies that are specific to aparticular domain of a gelsolin polypeptide is facilitated by generationof hybridomas that bind to the fragment of a gelsolin polypeptidepossessing such a domain. Thus, gelsolin binding agents which areantibodies that are specific for a desired domain within a gelsolinpolypeptide, or derivatives, fragments, analogs or homologs thereof, arealso provided herein.

The present invention further includes antibodies which areanti-idiotypic to the binding agents of the present invention. Thebinding agents of the present invention can be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific binding agentscan be specific for different epitopes of a gelsolin polypeptide of thepresent invention or can be specific for both a gelsolin polypeptide ofthe present invention as well as for heterologous compositions, such asa heterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893,5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol.148:1547-1553 (1992). The binding agents of the invention can be fromany animal origin, including birds and mammals. For example, the bindingagents may be from human, marine, rabbit, goat, guinea pig, camel,horse, or chicken.

The binding agents of the present invention can be used either alone orin combination with other compositions. For example, the gelsolinbinding agents of the invention can be used in combination with one ormore anti-gelsolin antibodies known in the art, e.g., but not limited toantibody GS-2C4. (Sigma-Aldrich, Cat. No. G4896; Afify and Werness.Appl. Immunohistochem. 6:30 (1998).)

The gelsolin binding agents of the present invention can further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, gelsolin binding agents of the present invention can berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

B. Methods of Preparing a Gelsolin-Binding Agents of the Invention

General Overview. Techniques for generating binding agents directed totarget polypeptides are well known to those skilled in the art. Examplesof such techniques include, but are not limited to, e.g., thoseinvolving display libraries, xeno or humab mice, hybridomas, and thelike. Target polypeptides within the scope of the present inventioninclude any polypeptide or polypeptide derivative which is capable ofexhibiting antigenicity. Examples include, but are not limited to,gelsolin and fragments thereof.

It should be understood that not only are naturally-occurring antibodiessuitable as binding agents for use in accordance with the presentdisclosure, but recombinantly engineered antibodies and antibodyfragments, e.g., antibody-related polypeptides, which are directed togelsolin polypeptide and fragments thereof are also suitable.

Binding agents, e.g., anti-gelsolin antibodies, that can be subjected tothe techniques set forth herein include monoclonal and polyclonalantibodies, and antibody fragments such as Fab, Fab′, F(ab′)₂, Fd, scFv,diabodies, antibody light chains, antibody heavy chains and/or antibodyfragments. Methods useful for the high yield production of antibodyFv-containing polypeptides, e.g., Fab′ and F(ab′)₂ antibody fragmentshave been described. See U.S. Pat. No. 5,648,237.

Generally, a binding agent is obtained from an originating species. Moreparticularly, the nucleic acid or amino acid sequence of the variableportion of the light chain, heavy chain or both, of an originatingspecies antibody having specificity for a target polypeptide antigen isobtained. Originating species is any species which was useful togenerate the binding agent of the invention or library of bindingagents, e.g., rat, mice, rabbit, chicken, monkey, human, and the like.

In some embodiments, gelsolin binding agents are anti-gelsolinantibodies. Phage or phagemid display technologies are useful techniquesto derive the binding agents of the present invention. Anti-gelsolinantibodies useful in the present invention are “human antibodies,”(e.g., antibodies isolated from a human) or “human sequence antibodies.”Human antibodies can be made by a variety of methods known in the artincluding phage display methods. See also, U.S. Pat. Nos. 4,444,887,4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO 98/50433, WO98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.Methods useful for the identification of nucleic acid sequences encodingmembers of multimeric polypeptide complex by screening polyphageparticles have been described. Rudert et al., U.S. Pat. No. 6,667,150.Also, recombinant immunoglobulins can be produced. Cabilly, U.S. Pat.No. 4,816,567; Cabilly et al., U.S. Pat. No. 6,331,415 and Queen et al.,Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989). Techniques forgenerating and cloning monoclonal antibodies are well known to thoseskilled in the art. The gelsolin binding agents of the inventionsuitably have a high immunoreactivity, that is, percentages ofantibodies molecules that are correctly folded so that they canspecifically bind their target antigen. Expression of sequences encodingbinding agents, e.g., antibodies of the invention, can be carried out inE. coli as described below. Such expression usually results inimmunoreactivity of at least 80%, 90%, 95% or 99%.

Certain truncations of these proteins or genes perform the regulatory orenzymatic functions of the full sequence protein or gene. For example,the nucleic acid sequences coding therefore can be altered bysubstitutions, additions, deletions or multimeric expression thatprovide for functionally equivalent proteins or genes. Due to thedegeneracy of nucleic acid coding sequences, other sequences whichencode substantially the same amino acid sequences as those of thenaturally occurring proteins may be used in the practice of the presentinvention. These include, but are not limited to, nucleic acid sequencesincluding all or portions of the nucleic acid sequences encoding theabove polypeptides, which are altered by the substitution of differentcodons that encode a functionally equivalent amino acid residue withinthe sequence, thus producing a silent change. It is appreciated that thenucleotide sequence of an immunoglobulin according to the presentinvention tolerates sequence homology variations of up to 25% ascalculated by standard methods (“Current Methods in Sequence Comparisonand Analysis,” Macromolecule Sequencing and Synthesis, Selected Methodsand Applications, Alan R Liss, Inc., 127-149, 1998) so long as such avariant forms an operative antibody which recognizes gelsolin orgelsolin-like polypeptides. For example, one or more amino acid residueswithin a polypeptide sequence can be substituted by another amino acidof a similar polarity which acts as a functional equivalent, resultingin a silent alteration. Substitutes for an amino acid within thesequence may be selected from other members of the class to which theamino acid belongs. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Also included within the scopeof the present invention are proteins or fragments or derivativesthereof which are differentially modified during or after translation,e.g., by glycosylation, protolytic cleavage, linkage to an antibodymolecule or other cellular ligands, etc. Additionally, an inhibitorencoding nucleic acid sequence can be mutated in vitro or in vivo tocreate and/or destroy translation, initiation, and/or terminationsequences or to create variations in coding regions and/or form newrestriction endonuclease sites or destroy pre-existing ones, tofacilitate further in vitro modification. Any technique for mutagenesisknown in the art can be used, including but not limited to in vitro sitedirected mutagenesis, J. Biol. Chem. 253:6551 (1978), use of Tab linkers(Pharmacia), and the like.

Preparation of Polyclonal Antisera and Immunogens.

Methods of generating antibodies or antibody fragments of the inventiontypically include immunizing a subject (generally a non-human subjectsuch as a mouse or rabbit) with a purified gelsolin or gelsolin-likepolypeptide or homolog or fragment thereof or with a cell expressing thegelsolin or gelsolin-like polypeptide or homolog or fragment thereof.Any immunogenic portion of the gelsolin polypeptide can be employed asthe immunogen. An appropriate immunogenic preparation can contain, e.g.,a recombinantly-expressed gelsolin polypeptide or achemically-synthesized gelsolin polypeptide. An isolated gelsolinpolypeptide, or a portion or fragment thereof, can be used as animmunogen to generate a gelsolin binding agent that binds to thegelsolin polypeptide, or a portion or fragment using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthgelsolin polypeptide can be used or, alternatively, the inventionprovides for the use of the gelsolin polypeptide fragments asimmunogens. The gelsolin polypeptide comprises at least four amino acidresidues of the amino acid sequence shown in SEQ ID NO.: 1, andencompasses an epitope of the gelsolin polypeptide such that an antibodyraised against the peptide forms a specific immune complex with thegelsolin polypeptide. The antigenic peptide may comprise at least 5, 8,10, 15, 20, or 30 amino acid residues. Longer antigenic peptides aresometimes preferable over shorter antigenic peptides, depending on useand according to methods well known to those skilled in the art.Typically, the immunogen will be at least about 8 amino acyl residues inlength, or at least about 10 acyl residues in length. Multimers of agiven epitope are sometimes more effective than a monomer.

If needed, the immunogenicity of the gelsolin polypeptide (or fragmentthereof) can be increased by fusion or conjugation to a hapten such askeyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptensare known in the art. One can also combine the gelsolin polypeptide witha conventional adjuvant such as Freund's complete or incomplete adjuvantto increase the subject's immune reaction to the polypeptide. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), human adjuvants such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory compounds. Thesetechniques are standard in the art.

For convenience, immune responses are often described in the presentinvention as being either “primary” or “secondary” immune responses. Aprimary immune response, which is also described as a “protective”immune response, refers to an immune response produced in an individualas a result of some initial exposure (e.g., the initial “immunization”)to a particular antigen, e.g., a gelsolin polypeptide. Such animmunization can occur, e.g., as the result of some natural exposure tothe antigen (e.g., from initial infection by some pathogen that exhibitsor presents the antigen) or from antigen presented by cancer cells ofsome tumor in the individual (e.g., malignant melanoma). Alternatively,the immunization can occur as a result of vaccinating the individualwith a vaccine containing the antigen. For example, the vaccine can be agelsolin vaccine comprising one or more antigens from a gelsolinpolypeptide or a gelsolin-like polypeptide.

A primary immune response can become weakened or attenuated over timeand can even disappear or at least become so attenuated that it cannotbe detected. Accordingly, the present invention also relates to a“secondary” immune response, which is also described here as a “memoryimmune response.” The term secondary immune response refers to an immuneresponse elicited in an individual after a primary immune response hasalready been produced.

Thus, a secondary or immune response can be elicited, e.g., to enhancean existing immune response that has become weakened or attenuated, orto recreate a previous immune response that has either disappeared orcan no longer be detected. The secondary or memory immune response canbe either a humoral (antibody) response or a cellular response. Asecondary or memory humoral response occurs upon stimulation of memory Bcells that were generated at the first presentation of the antigen.Delayed type hypersensitivity (DTH) reactions are a type of cellularsecondary or memory immune response that are mediated by CD4⁺ cells. Afirst exposure to an antigen primes the immune system and additionalexposure(s) results in a DTH.

Following appropriate immunization, the gelsolin binding agent, e.g.,anti-gelsolin polyclonal antibody can be prepared from the subject'sserum. If desired, the antibody molecules directed against the gelsolinpolypeptide can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

Monoclonal Antibody.

In one embodiment, the binding agent is an anti-gelsolin monoclonalantibody. For example, in some embodiments, the anti-gelsolin monoclonalantibody may be a human or a mouse anti-gelsolin monoclonal antibody.For preparation of monoclonal antibodies directed towards a particulargelsolin polypeptide, or derivatives, fragments, analogs or homologsthereof, any technique that provides for the production of antibodymolecules by continuous cell line culture can be utilized. Suchtechniques include, but are not limited to, the hybridoma technique(see, e.g., Kohler & Milstein, Nature 256:495-497 (1975)); the triomatechnique; the human B-cell hybridoma technique (see, e.g., Kozbor, etal., Immunol. Today 4:72 (1983)) and the EBV hybridoma technique toproduce human monoclonal antibodies. (See, e.g., Cole, et al., In:Monoclonal Antibodies And Cancer Therapy, Alan R Liss, Inc., 77-96(1985).) Human monoclonal antibodies can be utilized in the practice ofthe invention and can be produced by using human hybridomas (see, e.g.,Cote, et al., Proc. Natl. Acad. Sci. USA 80:2026-2030 (1983)) or bytransforming human B-cells with Epstein Barr Virus in vitro. (See, e.g.,Cole, et al., In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss,Inc., 77-96 (1985).) For example, a population of nucleic acids thatencode regions of antibodies can be isolated. PCR utilizing primersderived from sequences encoding conserved regions of antibodies is usedto amplify sequences encoding portions of antibodies from the populationand then reconstruct DNAs encoding antibodies or fragments thereof, suchas variable domains, from the amplified sequences. Such amplifiedsequences also can be fused to DNAs encoding other proteins—e.g., abacteriophage coat, or a bacterial cell surface protein—for expressionand display of the fusion polypeptides on phage or bacteria. Amplifiedsequences can then be expressed and further selected or isolated based,e.g., on the affinity of the expressed antibody or fragment thereof foran antigen or epitope present on the gelsolin polypeptide.Alternatively, hybridomas expressing anti-gelsolin monoclonal antibodiescan be prepared by immunizing a subject and then isolating hybridomasfrom the subject's spleen using routine methods. See, e.g., Milstein etal., Galfre and Milstein, Methods Enzymol 73:3-46 (1981). Screening thehybridomas using standard methods will produce monoclonal antibodies ofvarying specificity (i.e., for different epitopes) and affinity. Aselected monoclonal antibody with the desired properties, e.g., gelsolinbinding, can be used as expressed by the hybridoma, it can be bound to amolecule such as polyethylene glycol (PEG) to alter its properties, or acDNA encoding it can be isolated, sequenced and manipulated in variousways. Synthetic dendromeric trees can be added a reactive amino acidside chains, e.g., lysine to enhance the immunogenic properties of thegelsolin polypeptide. Also, CPG-dinucleotide technique can be used toenhance the immunogenic properties of the gelsolin polypeptide. Othermanipulations include substituting or deleting particular amino acylresidues that contribute to instability of the antibody during storageor after administration to a subject, and affinity maturation techniquesto improve affinity of the antibody of the gelsolin polypeptide.

Hybridoma Technique.

In one embodiment, the binding agent of the invention is ananti-gelsolin monoclonal antibody produced by a hybridoma which includesa B cell obtained from a transgenic non-human animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell. Hybridomatechniques include those known in the art and taught in Harlow et al.,Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. 349 (1988); Hammerling et al., Monoclonal AntibodiesAnd T-Cell Hybridomas, 563-681 (1981). Other methods for producinghybridomas and monoclonal antibodies are well known to those of skill inthe art.

Phage Display Technique.

As noted above, the binding agents of the present invention can beproduced through the application of recombinant DNA and phage displaytechnology. For example, binding agents of the invention, e.g.,anti-gelsolin antibodies, can be prepared using various phage displaymethods known in the art. In phage display methods, functional antibodydomains are displayed on the surface of a phage particle which carriespolynucleotide sequences encoding them. Phage with a desired bindingproperty are selected from a repertoire or combinatorial antibodylibrary (e.g., human or murine) by selecting directly with antigen,typically antigen bound or captured to a solid surface or bead. Phageused in these methods are typically filamentous phage including fd andM13 with Fab, Fv or disulfide stabilized Fv antibody domains arerecombinantly fused to either the phage gene III or gene VIII protein.In addition, methods can be adapted for the construction of Fabexpression libraries (see, e.g., Huse, et al., Science 246:1275-1281,(1989)), to allow rapid and effective identification of monoclonal Fabfragments with the desired specificity for a gelsolin polypeptide, e.g.,a polypeptide or derivatives, fragments, analogs or homologs thereof.Other examples of phage display methods that can be used to make thebinding agents of the present invention include those disclosed inHuston et al., Proc. Natl. Acad. Sci. USA. 85:5879-5883 (1988);Chaudhary et al., Proc. Natl. Acad. Sci. USA. 87:1066-1070 (1990);Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J.Immunol. 24:952-958 (1994); Persic et al., Gene 187:9-18 (1997; Burtonet al., Advances in Immunology 57:191-280 (1994); PCT/GB91/01134; WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical ResearchCouncil et al.); WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO91/17271 (Affymax); and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods useful fordisplaying polypeptides on the surface of bacteriophage particles byattaching the polypeptides via disulfide bonds have been described byLohning, U.S. Pat. No. 6,753,136. As described in the above references,after phage selection, the antibody coding regions from the phage can beisolated and used to generate whole antibodies, including humanantibodies, or any other desired antigen binding fragment, and expressedin any desired host including mammalian cells, insect cells, plantcells, yeast, and bacteria. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in WO 92/22324;Mullinax et al., BioTechniques 12:864-869 (1992); and Sawai et al., AJRI34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988).

Generally, hybrid antibodies or hybrid antibody fragments that arecloned into a display vector can be selected against the appropriateantigen in order to identify variants that maintained good bindingactivity, because the antibody or antibody fragment will be present onthe surface of the phage or phagemid particle. See e.g., Barbas III etal., Phage Display, A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001). However, other vector formatscould be used for this process, such as cloning the antibody fragmentlibrary into a lytic phage vector (modified T7 or Lambda Zap systems)for selection and/or screening.

Expression of Recombinant Gelsolin-Binding Agent. As noted above, thebinding agents of the present invention can be produced through theapplication of recombinant DNA technology. Recombinant polynucleotideconstructs encoding a gelsolin binding agent of the present inventiontypically include an expression control sequence operably-linked to thecoding sequences of anti-gelsolin antibody chains, includingnaturally-associated or heterologous promoter regions. As such, anotheraspect of the invention includes vectors containing one or more nucleicacid sequences encoding a gelsolin binding agent of the presentinvention. For recombinant expression of one or more the polypeptides ofthe invention, the nucleic acid containing all or a portion of thenucleotide sequence encoding the gelsolin binding agent is inserted intoan appropriate cloning vector, or an expression vector (i.e., a vectorthat contains the necessary elements for the transcription andtranslation of the inserted polypeptide coding sequence) by recombinantDNA techniques well known in the art and as detailed below. Methods forproducing diverse populations of vectors have been described by Lerneret al., U.S. Pat. Nos. 6,291,160; 6,680,192.

In general, expression vectors useful in recombinant DNA techniques areoften in the form of plasmids. In the present specification, “plasmid”and “vector” can be used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors that are not technicallyplasmids, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. Such viral vectors permit infection of a subjectand expression in that subject of a compound. The expression controlsequences are typically eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences encoding the gelsolin binding agent, and the collection andpurification of the gelsolin binding agent, e.g., cross-reactinganti-gelsolin antibodies. See, generally, U.S. Application No.20020199213. These expression vectors are typically replicable in thehost organisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markers,e.g., ampicillin-resistance or hygromycin-resistance, to permitdetection of those cells transformed with the desired DNA sequences.Vectors can also encode signal peptide, e.g., pectate lyase, useful todirect the secretion of extracellular antibody fragments. See U.S. Pat.No. 5,576,195.

The recombinant expression vectors comprise a nucleic acid encoding acompound with gelsolin binding properties in a form suitable forexpression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression thatis operatively-linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably-linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, e.g., in Goeddel,Gene Expression Technology: Methods In Enzymology 185, Academic Press,San Diego, Calif. (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcell and those that direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. Typical regulatory sequences useful as promoters of recombinantpolypeptide expression (e.g., gelsolin binding agents), include, e.g.,but are not limited to, 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization. In one embodiment, a polynucleotideencoding a gelsolin binding agent of the invention is operably-linked toan ara B promoter and expressible in a host cell. See U.S. Pat. No.5,028,530. The expression vectors of the invention can be introducedinto host cells to thereby produce polypeptides or peptides, includingfusion polypeptides, encoded by nucleic acids as described herein (e.g.,gelsolin binding agents, etc.).

Another aspect of the invention pertains to gelsolin bindingagent-expressing host cells, which contain a nucleic acid encoding oneor more gelsolin binding agents. The recombinant expression vectors ofthe invention can be designed for expression of a gelsolin binding agentin prokaryotic or eukaryotic cells. For example, a gelsolin bindingagent can be expressed in bacterial cells such as Escherichia coli,insect cells (using baculovirus expression vectors), fungal cells, e.g.,yeast, yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel, Gene Expression Technology: Methods In Enzymology185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.Methods useful for the preparation screening of polypeptides havingpredetermined property, e.g., gelsolin binding agents, via expression ofstochastically generated polynucleotide sequences has been described.See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483;5,824,514; 5,976,862; 6,492,107; 6,569,641.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion polypeptides.Fusion vectors add a number of amino acids to a polypeptide encodedtherein, usually to the amino terminus of the recombinant polypeptide.Such fusion vectors typically serve three purposes: (i) to increaseexpression of recombinant polypeptide; (ii) to increase the solubilityof the recombinant polypeptide; and (iii) to aid in the purification ofthe recombinant polypeptide by acting as a ligand in affinitypurification. Often, in fusion expression vectors, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant polypeptide to enable separation of the recombinantpolypeptide from the fusion moiety subsequent to purification of thefusion polypeptide. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding polypeptide, or polypeptide A,respectively, to the target recombinant polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., Gene 69:301-315 (1988) and pET 11d (Studieret al., Gene Expression Technology: Methods In Enzymology 185, AcademicPress, San Diego, Calif. 60-89 (1990)). Methods for targeted assembly ofdistinct active peptide or protein domains to yield multifunctionalpolypeptides via polypeptide fusion has been described by Pack et al.,U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy to maximizerecombinant polypeptide expression, e.g., a gelsolin binding agent, inE. coli is to express the polypeptide in host bacteria with an impairedcapacity to proteolytically cleave the recombinant polypeptide. See,e.g., Gottesman, Gene Expression Technology: Methods In Enzymology 185,Academic Press, San Diego, Calif. 119-128 (1990). Another strategy is toalter the nucleic acid sequence of the nucleic acid to be inserted intoan expression vector so that the individual codons for each amino acidare those preferentially utilized in the expression host, e.g., E. coli.(See, e.g., Wada, et al., Nucl. Acids Res. 20:2111-2118 (1992).) Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

In another embodiment, the gelsolin binding agent expression vector is ayeast expression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933-943 (1982)),pJRY88 (Schultz et al., Gene 54:113-123 (1987)), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). Alternatively, a gelsolin binding agent can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of polypeptides, e.g., gelsolin binding agents,in cultured insect cells (e.g., SF9 cells), include the pAc series(Smith, et al., Mol. Cell. Biol. 3:2156-2165 (1983)), and the pVL series(Lucklow and Summers, Virology 170:31-39 (1989)).

In yet another embodiment, a nucleic acid encoding a gelsolin bindingagent of the invention is expressed in mammalian cells using a mammalianexpression vector. Examples of mammalian expression vectors include,e.g., but are not limited to, pCDM8 (Seed, Nature 329:840 (1987)), andpMT2PC (Kaufman, et al., EMBO J. 6:187-195 (1987)). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells useful for expression of the gelsolinbinding agents of the present invention. See, e.g., Chapters 16 and 17of Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1989).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,Genes Dev. 1:268-277 (1987)), lymphoid-specific promoters (Calame andEaton, Adv. Immunol. 43:235-275 (1988)), in particular promoters of Tcell receptors (Winoto and Baltimore, EMBO J. 8:729-733 (1989)), andimmunoglobulins (Banerji, et al., Cell 33:729-740 (1993); Queen andBaltimore, Cell 33:741-748 (1983)), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA86:5473-5477 (1989)), pancreas-specific promoters (Edlund, et al.,Science 230:912-916 (1985)), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, Science249:374-379 (1990)), and the α-fetoprotein promoter (Campes andTilghman, Genes Dev. 3:537-546 (1989)).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, agelsolin binding agent can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells. Mammalian cells are onehost for expressing nucleotide segments encoding immunoglobulins orfragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers,New York (1987)). A number of suitable host cell lines capable ofsecreting intact heterologous proteins have been developed in the art,and include Chinese hamster ovary (CHO) cell lines, various COS celllines, HeLa cells, L cells and myeloma cell lines. Preferably, the cellsare nonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Queen et al., Immunol. Rev. 89:49(1986). Preferred expression control sequences are promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. Co et al., J Immunol. 148:1149 (1992).Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation, biolistics or viral-based transfectioncan be used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., Molecular Cloning: A Laboratory Manual). Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989)), and other laboratory manuals. The vectors containing theDNA segments of interest can be transferred into the host cell by wellknown methods, depending on the type of cellular host.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding the gelsolin binding agent or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell that includes a gelsolin binding agent of the presentinvention, such as a prokaryotic or eukaryotic host cell in culture, canbe used to produce (i.e., express) recombinant gelsolin binding agent.In one embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding thegelsolin binding agent has been introduced) in a suitable medium suchthat the gelsolin binding agent is produced. In another embodiment, themethod further comprises the step of isolating the gelsolin bindingagent from the medium or the host cell. Once expressed, collections ofthe gelsolin binding agents, e.g., the anti-gelsolin antibodies or theanti-gelsolin antibody-related polypeptides are purified from culturemedia and host cells. The gelsolin binding agents can be purifiedaccording to standard procedures of the art, including HPLCpurification, column chromatography, gel electrophoresis and the like.In one embodiment, the gelsolin binding agent is produced in a hostorganism by the method of Boss et al., U.S. Pat. No. 4,816,397. Usually,anti-gelsolin antibody chains are expressed with signal sequences andare thus released to the culture media. However, if the anti-gelsolinantibody chains are not naturally secreted by host cells, theanti-gelsolin antibody chains can be released by treatment with milddetergent. Purification of recombinant polypeptides is well known in theart and include ammonium sulfate precipitation, affinity chromatographypurification technique, column chromatography, ion exchange purificationtechnique, gel electrophoresis and the like (see generally Scopes,Protein Purification (Springer-Verlag, N.Y. (1982)).

Polynucleotides encoding gelsolin binding agents, e.g., theanti-gelsolin antibody coding sequences, can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal. See, e.g.,U.S. Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenesinclude coding sequences for light and/or heavy chains in operablelinkage with a promoter and enhancer from a mammary gland specific gene,such as casein or β-lactoglobulin. For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

Single Chain Antibodies.

In one embodiment, the binding agent of the invention is a single chainanti-gelsolin antibody. According to the invention, techniques can beadapted for the production of single-chain antibodies specific to agelsolin polypeptide (see, e.g., U.S. Pat. No. 4,946,778). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesof the invention include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu,L. et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerraet al., Science 240:1038-1040 (1988).

Chimeric and Humanized Antibodies.

In one embodiment, the binding agent of the invention is a chimericanti-gelsolin antibody. In one embodiment, the binding agent of theinvention is a humanized anti-gelsolin antibody. In one embodiment ofthe invention, the donor and acceptor antibodies are monoclonalantibodies from different species. For example, the acceptor antibody isa human antibody (to minimize its antigenicity in a human), in whichcase the resulting CDR-grafted antibody is termed a “humanized”antibody.

Recombinant anti-gelsolin antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions, canbe made using standard recombinant DNA techniques, and are within thescope of the invention. For some uses, including in vivo use of thebinding agent of the invention in humans as well as use of these agentsin vitro detection assays, it is preferable to use chimeric, humanized,or human anti-gelsolin antibodies. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art. Such useful methods include, e.g., but are not limitedto, methods described in International Application No. PCT/US86/02269;U.S. Pat. No. 5,225,539; European Patent No. 184187, European Patent No.171496; European Patent No. 173494; PCT International Publication No. WO86/01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No.125023; Better, et al., Science 240:1041-1043 (1988); Liu, et al., Proc.Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, et al., J. Immunol.139:3521-3526 (1987); Sun, et al., Proc. Natl. Acad. Sci. USA 84:214-218(1987); Nishimura, et al., Cancer Res. 47:999-1005 91987); Wood, et al.,Nature 314:446-449 (1985); Shaw, et al., J. Natl. Cancer Inst.80:1553-1559 (1988); Morrison, Science 229:1202-1207 (1985); Oi, et al.,BioTechniques 4:214 (1986); Jones, et al., Nature 321:552-525 (1986);Verhoeyan, et al., Science 239:1534 1988); Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J.Immunol. Methods, 125:191-202 (1989); U.S. Pat. No. 5,807,715; andBeidler, et al., J. Immunol. 141:4053-4060 (1988). For example,antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101;5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology, 28:489-498(1991); Studnicka et al., Protein Engineering 7:805-814 (1994); Roguskaet al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No.5,565,332). In one embodiment, a cDNA encoding a murine anti-gelsolinmonoclonal antibody is digested with a restriction enzyme selectedspecifically to remove the sequence encoding the Fc constant region, andthe equivalent portion of a cDNA encoding a human Fc constant region issubstituted (see Robinson et al., PCT/US86/02269; Akira et al., EuropeanPatent Application 184,187; Taniguchi, European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly etal., European Patent Application 125,023; Better et al., Science240:1041-1043 (1988); Liu et al., Proc. Natl. Acad. Sci. USA84:3439-3443 (1987); Liu et al., J Immunol 139:3521-3526 (1987); Sun etal., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura et al.,Cancer Res 47:999-1005 (1987); Wood et al., Nature 314:446-449 (1985);and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559 (1988); U.S. Pat.No. 6,180,370; U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484;6,828,422.

In one embodiment, the present invention allows the construction ofhumanized anti-gelsolin antibodies that are unlikely to induce a humananti-mouse antibody (hereinafter referred to as “HAMA”) response, whilestill having an effective antibody effector function. As used herein,the terms “human” and “humanized”, in relation to antibodies, relate toany antibody which is expected to elicit a therapeutically tolerableweak immunogenic response in a human subject. In one embodiment, thepresent invention provides for a humanized gelsolin antibodies, heavyand light chain immunoglobulins.

CDR Antibodies.

In one embodiment, the binding agent of the invention is ananti-gelsolin CDR antibody. Generally the donor and acceptor antibodiesused to generate the anti-gelsolin CDR antibody are monoclonalantibodies from different species; typically the acceptor antibody is ahuman antibody (to minimize its antigenicity in a human), in which casethe resulting CDR-grafted antibody is termed a “humanized” antibody. Thegraft may be of a single CDR (or even a portion of a single CDR) withina single V_(H) or V_(L) of the acceptor antibody, or can be of multipleCDRs (or portions thereof) within one or both of the V_(H) and V_(L).Frequently all three CDRs in all variable domains of the acceptorantibody will be replaced with the corresponding donor CDRs, though oneneed replace only as many as necessary to permit adequate binding of theresulting CDR-grafted antibody to MetAp3. Methods for generatingCDR-grafted and humanized antibodies are taught by Queen et al. U.S.Pat. No. 5,585,089, U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762;and Winter U.S. Pat. No. 5,225,539; and EP 0682040. Methods useful toprepare V_(H) and V_(L) polypeptides are taught by Winter et al., U.S.Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP0368684; EPO451216; EP0120694.

After selecting suitable framework region candidates from the samefamily and/or the same family member, either or both the heavy and lightchain variable regions are produced by grafting the CDRs from theoriginating species into the hybrid framework regions. Assembly ofhybrid antibodies or hybrid antibody fragments having hybrid variablechain regions with regard to either of the above aspects can beaccomplished using conventional methods known to those skilled in theart. For example, DNA sequences encoding the hybrid variable domainsdescribed herein (i.e., frameworks based on the target species and CDRsfrom the originating species) can be produced by oligonucleotidesynthesis and/or PCR. The nucleic acid encoding CDR regions can also beisolated from the originating species antibodies using suitablerestriction enzymes and ligated into the target species framework byligating with suitable ligation enzymes. Alternatively, the frameworkregions of the variable chains of the originating species antibody canbe changed by site-directed mutagenesis.

Since the hybrids are constructed from choices among multiple candidatescorresponding to each framework region, there exist many combinations ofsequences which are amenable to construction in accordance with theprinciples described herein. Accordingly, libraries of hybrids can beassembled having members with different combinations of individualframework regions. Such libraries can be electronic database collectionsof sequences or physical collections of hybrids.

This process typically does not alter the acceptor antibody's FRsflanking the grafted CDRs. However, one skilled in the art can sometimesimprove antigen binding affinity of the resulting anti-gelsolin CDRgrafted antibody by replacing certain residues of a given FR to make theFR more similar to the corresponding FR of the donor antibody. Preferredlocations of the substitutions include amino acid residues adjacent tothe CDR, or which are capable of interacting with a CDR. (See, e.g.,U.S. Pat. No. 5,585,089, especially columns 12-16). Or one skilled inthe art can start with the donor FR and modify it to be more similar tothe acceptor FR or a human consensus FR. Techniques for making thesemodifications are known in the art. Particularly if the resulting FRfits a human consensus FR for that position, or is at least 90% or moreidentical to such a consensus FR, doing so may not increase theantigenicity of the resulting modified anti-gelsolin CDR antibodysignificantly compared to the same antibody with a fully human FR.

Fusion Proteins.

In one embodiment, the binding agent of the invention is a fusionprotein. The gelsolin binding agents of the present invention, whenfused to a second protein, can be used as an antigenic tag. Examples ofdomains that can be fused to polypeptides include not only heterologoussignal sequences, but also other heterologous functional regions. Thefusion does not necessarily need to be direct, but can occur throughlinker sequences. Moreover, fusion proteins of the present invention canalso be engineered to improve characteristics of the gelsolin bindingagent. For instance, a region of additional amino acids, particularlycharged amino acids, can be added to the N-terminus of the gelsolinbinding agent to improve stability and persistence during purificationfrom the host cell or subsequent handling and storage. Also, peptidemoieties can be added to the gelsolin binding agent to facilitatepurification. Such regions can be removed prior to final preparation ofthe gelsolin binding agent. The addition of peptide moieties tofacilitate handling of polypeptides are familiar and routine techniquesin the art. The gelsolin binding agent of the invention can be fused tomarker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., Chatsworth, Calif.), among others, many of whichare commercially available. As described in Gentz et al., Proc. Natl.Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine providesfor convenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. Wilson et al., Cell 37:767(1984).

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention. Also, thefusion protein can show an increased half-life in vivo.

Fusion proteins having disulfide-linked dimeric structures (due to theIgG) can be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).

Similarly, EP-A-0 464 533 discloses fusion proteins comprising variousportions of constant region of immunoglobulin molecules together withanother human protein or part thereof. In many cases, the Fc part in afusion protein is beneficial in therapy and diagnosis, and thus canresult in, e.g., improved pharmacokinetic properties. See EP-A 0232 262.Alternatively, deleting the Fc part after the fusion protein has beenexpressed, detected, and purified, would be desired. For example, the Fcportion can hinder therapy and diagnosis if the fusion protein is usedas an antigen for immunizations. In drug discovery, e.g., humanproteins, such as hIL-5, have been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists ofhIL-5. Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johansonet al., J. Biol. Chem., 270:9459-9471 (1995).

Labeled Gelsolin-Binding Agent.

In one embodiment, the gelsolin binding agent of the present inventionis coupled with a label moiety, i.e., detectable group. The particularlabel or detectable group conjugated to the gelsolin binding agent ofthe invention is not a critical aspect of the invention, so long as itdoes not significantly interfere with the specific binding of thegelsolin binding agent of the present invention to the gelsolinpolypeptide or the gelsolin-like polypeptide. The detectable group canbe any material having a detectable physical or chemical property. Suchdetectable labels have been well-developed in the field of immunoassaysand imaging, in general, most any label useful in such methods can beapplied to the present invention. Thus, a label is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include magnetic beads (e.g., Dynabeads™) fluorescent dyes(e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like),radiolabels (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹²¹I, ¹³¹I, ¹¹²In, ⁹⁹mTc), otherimaging agents such as microbubbles (for ultrasound imaging), ¹⁸F, ¹¹C,¹⁵O, (for Positron emission tomography), ⁹⁹mTC, ¹¹¹In (for Single photonemission tomography), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and calorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, and the like) beads. Patents thatdescribed the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241,each incorporated herein by reference in their entirety and for allpurposes. See also Handbook of Fluorescent Probes and Research Chemicals(6^(th) Ed., Molecular Probes, Inc., Eugene Oreg. (1996)).

The label can be coupled directly or indirectly to the desired componentof an assay according to methods well known in the art. As indicatedabove, a wide variety of labels can be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine,and cortisol, it can be used in conjunction with the labeled,naturally-occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody, e.g., ananti-gelsolin antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds useful as labelingmoieties, include, but are not limited to, e.g., fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, andthe like. Chemiluminescent compounds useful as labelling moieties,include, but are not limited to, e.g., luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal-producing systems which can be used, see, U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels can bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels can be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies, e.g., the anti-gelsolin antibodies. In this case,antigen-coated particles are agglutinated by samples comprising thetarget antibodies. In this format, none of the components need belabeled and the presence of the target antibody is detected by simplevisual inspection.

C. Identifying and Characterizing the Gelsolin-Binding Agents of theInvention

Methods for Identifying and/or Screening the Binding Agents of theInvention.

Methods useful to identify and screen the binding agents, e.g.,anti-gelsolin antibodies and anti-gelsolin antibody-relatedpolypeptides, that possess the desired specificity to the gelsolinpolypeptide include any immunologically-mediated techniques known withinthe art. Components of an immune response can be detected in vitro byvarious methods that are well known to those of ordinary skill in theart. For example, (1) cytotoxic T lymphocytes can be incubated withradioactively labeled target cells and the lysis of these target cellsdetected by the release of radioactivity; (2) helper T lymphocytes canbe incubated with antigens and antigen presenting cells and thesynthesis and secretion of cytokines measured by standard methods(Windhagen A; et al., Immunity, 2:373-80 (1995)); (3) antigen presentingcells can be incubated with whole protein antigen and the presentationof that antigen on MHC detected by either T lymphocyte activation assaysor biophysical methods (Harding et al., Proc. Natl. Acad. Sci.,86:4230-4 (1989)); (4) mast cells can be incubated with reagents thatcross-link their Fc-epsilon receptors and histamine release measured byenzyme immunoassay (Siraganian et al., TIPS, 4:432-437 (1983)); and (5)enzyme-linked immunosorbent assay (ELISA).

Similarly, products of an immune response in either a model organism(e.g., mouse) or a human subject can also be detected by various methodsthat are well known to those of ordinary skill in the art. For example,(1) the production of antibodies in response to vaccination can bereadily detected by standard methods currently used in clinicallaboratories, e.g., an ELISA; (2) the migration of immune cells to sitesof inflammation can be detected by scratching the surface of skin andplacing a sterile container to capture the migrating cells over scratchsite (Peters et al., Blood, 72:1310-5 (1988)); (3) the proliferation ofperipheral blood mononuclear cells in response to mitogens or mixedlymphocyte reaction can be measured using ³H-thymidine; (4) thephagocytic capacity of granulocytes, macrophages, and other phagocytesin PBMCs can be measured by placing PMBCs in wells together with labeledparticles (Peters et al., Blood, 72:1310-5 (1988)); and (5) thedifferentiation of immune system cells can be measured by labeling PBMCswith antibodies to CD molecules such as CD4 and CD8 and measuring thefraction of the PBMCs expressing these markers.

In one embodiment, gelsolin binding agents of the invention are selectedusing display of candidate binding agents on the surface of replicablegenetic packages. See, e.g., U.S. Pat. Nos. 5,514,548; 5,837,500;5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844306. Methods useful for producing/selecting a filamentous bacteriophageparticle containing a phagemid genome encoding for a binding moleculewith a desired specificity has been described. See, e.g., EP 774 511;U.S. Pat. No. 5,871,907; U.S. Pat. No. 5,969,108; U.S. Pat. No.6,225,447; U.S. Pat. No. 6,291,650; U.S. Pat. No. 6,492,160.

In one embodiment, gelsolin binding agents of the invention are selectedusing display of candidate binding agents on the surface of a yeast hostcell. Methods useful for the isolation of scFv polypeptides by yeastsurface display have been described by Kieke et al., Protein Eng.,10(11):1303-10 (1997).

In one embodiment, gelsolin binding agents of the invention are selectedusing ribosome display. Methods useful for identifying ligands inpeptide libraries using ribosome display have been described byMattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-26 (1994); andHanes et al., Proc. Natl. Acad. Sci. USA 94:4937-42 (1997).

In one embodiment, gelsolin binding agents of the invention are selectedusing tRNA display of candidate binding agents. Methods useful for invitro selection of ligands using tRNA display have been described byMerryman et al., Chem. Biol. 9:741-46 (2002).

In one embodiment, gelsolin binding agents of the invention are selectedusing RNA display. Methods useful for selecting peptides and proteinsusing RNA display libraries have been described by Roberts et al. Proc.Natl. Acad. Sci. USA, 94:12297-302 (1997); and Nemoto et al., FEBSLett., 414:405-8 (1997). Methods useful for selecting peptides andproteins using unnatural RNA display libraries have been described byFrankel et al., Curr. Opin. Struct. Biol., 13:506-12 (2003).

In one embodiment, gelsolin binding agents of the invention areexpressed in the periplasm of gram negative bacteria and mixed withlabeled gelsolin polypeptide. See WO 02/34886. In clones expressingrecombinant polypeptides with affinity for the gelsolin polypeptide, theconcentration of the labeled gelsolin polypeptide bound to the bindingagents is increased and allows the cells to be isolated from the rest ofthe library as described in Harvey et al., Proc. Natl. Acad. Sci.22:9193-98 (2004) and U.S. Pat. Publication No. 2004/0058403.

After selection of the desired gelsolin binding agent, it iscontemplated that it can be produced in large volume by any techniqueknown to those skilled in the art, e.g., prokaryotic or eukaryotic cellexpression and the like. The gelsolin binding agents which are, e.g.,but not limited to, anti-gelsolin hybrid antibodies or fragments can beproduced by using conventional techniques to construct an expressionvector that encodes an antibody heavy chain in which the CDRs and, ifnecessary, a minimal portion of the variable region framework, that arerequired to retain original species antibody binding specificity (asengineered according to the techniques described herein) are derivedfrom the originating species antibody and the remainder of the antibodyis derived from a target species immunoglobulin which can be manipulatedas described herein, thereby producing a vector for the expression of ahybrid antibody heavy chain.

Measurement of Gelsolin Binding.

In one embodiment, a gelsolin binding assay refers to an assay formatwherein a gelsolin polypeptide and a gelsolin binding agent are mixedunder conditions suitable for binding between the gelsolin orgelsolin-like polypeptide and the gelsolin binding agent and assessingthe amount of binding between the gelsolin or gelsolin-like polypeptideand the gelsolin binding agent. The amount of binding is compared with asuitable control, which can be the amount of binding in the absence ofthe gelsolin polypeptide, the amount of the binding in the presence ofnon-specific immunoglobulin composition, or both. The amount of bindingcan be assessed by any suitable method. Binding assay methods include,e.g., ELISA, radioimmunoassays, scintillation proximity assays,fluorescence energy transfer assays, liquid chromatography, membranefiltration assays, and the like. Biophysical assays for the directmeasurement of gelsolin polypeptide binding to gelsolin binding agentsare, e.g., nuclear magnetic resonance, fluorescence, fluorescencepolarization, surface plasmon resonance (BIACOR chips) and the like.Specific binding is determined by standard assays known in the art,e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR,NMR (2D-NMR), mass spectroscopy and the like. If the specific binding ofa candidate gelsolin binding agent is at least 1 percent greater thanthe binding observed in the absence of the candidate gelsolin bindingagent, the candidate gelsolin binding agent is useful as a gelsolinbinding agent of the invention.

Co-crystals of the gelsolin polypeptides and the gelsolin binding agentsare also provided by the present invention as a method of determiningmolecular interactions. Conditions suitable for binding between thegelsolin binding agent and a gelsolin polypeptide will depend on thecompound and its ligand and can be readily determined by one of ordinaryskill in the art.

II. Uses of the Gelsolin Binding Agents of the Invention

General. The binding agents of the invention are useful in methods knownin the art relating to the localization and/or quantitation of agelsolin or gelsolin-like polypeptide (e.g., for use in measuring levelsof the gelsolin or gelsolin-like polypeptide within appropriatephysiological samples, for use in diagnostic methods, for use in imagingthe polypeptide, and the like). Binding agents of the invention areuseful to isolate a gelsolin polypeptide by standard techniques, such asaffinity chromatography or immunoprecipitation. A gelsolin binding agentof the invention can facilitate the purification of naturalimmunoreactive gelsolin polypeptides or gelsolin-like polypeptides frombiological samples, e.g., mammalian sera, urine, or cells as well asrecombinantly-produced immunoreactive gelsolin polypeptides orgelsolin-like polypeptides expressed in a host system. Moreover,gelsolin binding agent can be used to detect an immunoreactive gelsolinpolypeptide or a gelsolin-like polypeptide (e.g., in plasma, urine, acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the immunoreactive polypeptide. Thegelsolin binding agents of the invention can be used diagnostically tomonitor immunoreactive gelsolin and/or gelsolin-like polypeptide levelsin tissue as part of a clinical testing procedure, e.g., to determinethe efficacy of a given treatment regimen. As noted above, the detectioncan be facilitated by coupling (i.e., physically linking) the gelsolinbinding agent of the invention to a detectable substance.

Detection of Gelsolin Polypeptide.

An exemplary method for detecting the presence or absence of a gelsolinpolypeptide or a gelsolin-like polypeptide in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a gelsolin binding agent of theinvention capable of detecting a gelsolin polypeptide or a gelsolin-likepolypeptide such that the presence of a gelsolin polypeptide or agelsolin-like polypeptide is detected in the biological sample. Anexample of a gelsolin binding agent is an antibody raised against SEQ IDNO.: 1 or a homlog or fragment thereof, capable of binding to a gelsolinpolypeptide (a gelsolin polypeptide fragment) or a gelsolin-likepolypeptide. The gelsolin binding agent may be labeled. The term“labeled,” with regard to the binding agent is intended to encompassdirect labeling of the binding agent by coupling (i.e., physicallylinking) a detectable substance to the binding agent, as well asindirect labeling of the binding agent by reactivity with anothercompound that is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently-labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently-labeled streptavidin.

In suitable embodiments, a urine sample is assayed for a gelsolinpolypeptide or gelsolin-like polypeptide. While not wishing to belimited by theory, urine gelsolin or gelsolin-like polypeptides may be aproduct of the utilization of plasma gelsolin. Plasma gelsolin is a 90kDa polypeptide and has a critical biological function in binding andclearance of actin and other toxic substances released from injuredcells. Lower levels of plasma gelsolin are associated with manydiseases, suggesting that depletion of plasma gelsolin occurs during thedisease process. After binding to actin, full-length gelsolin may becleared by the kidney. Thus, urine gelsolin fragments represent abiomarker for in vivo utilization of plasma gelsolin.

The detection method of the invention can be used to detect a gelsolinpolypeptide or a gelsolin-like polypeptide in a biological sample invitro as well as in vivo. In vitro techniques for detection of agelsolin polypeptide or a gelsolin-like polypeptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, radioimmunoassay, and immunofluorescence.Furthermore, in vivo techniques for detection of a gelsolin polypeptideor a gelsolin-like polypeptide include introducing into a subject alabeled gelsolin binding agent, e.g., an anti-gelsolin antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. In one embodiment, the biological sample containspolypeptide molecules from the test subject.

Immunoassay and Imaging.

A gelsolin binding agent of the present invention can be used to assaygelsolin polypeptide levels or gelsolin-like polypeptide levels in abiological sample (e.g., human plasma) using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. Jalkanen, M. et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096(1987. Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes or other radioactive agent, such as iodine(¹²¹I, ¹²¹I, ¹³¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, such asfluorescein and rhodamine, and biotin.

In addition to assaying gelsolin polypeptide levels or gelsolin-likepolypeptide levels in a biological sample, gelsolin polypeptide levelsor gelsolin-like polypeptide levels can also be detected in vivo byimaging. A gelsolin binding agent, e.g., an anti-gelsolin antibodylabels or markers for in vivo imaging of the gelsolin polypeptide levelsor the gelsolin-like polypeptide include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which can be incorporated into the gelsolin binding agent bylabeling of nutrients for the relevant scFv clone.

A gelsolin binding agent which has been labeled with an appropriatedetectable imaging moiety, such as a radioisotope (e.g., ¹³¹I, ¹¹²In,^(99m)Tc), a radio-opaque substance, or a material detectable by nuclearmagnetic resonance, is introduced (e.g., parenterally, subcutaneously,or intraperitoneally) into the subject. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ^(99m)Tc. The labeled gelsolin binding agent will thenpreferentially accumulate at the location of cells which contain thespecific target polypeptide. For example, in vivo tumor imaging isdescribed in S. W. Burchiel et al., Tumor Imaging: The RadiochemicalDetection of Cancer 13 (1982).

Thus, the invention provides a diagnostic method of a medical condition,which involves: (a) assaying the expression of a polypeptide bymeasuring binding of a gelsolin binding agent of the present inventionin cells or body fluid of an individual; (b) comparing the amount ofprotein with a standard, whereby an increase or decrease in the assayedpolypeptide compared to the standard level is indicative of a medicalcondition.

Affinity Purification.

The gelsolin binding agents of the present invention may be used topurify immunoreacitve gelsolin (e.g., native plasma gelsolin) from asample. In some embodiments, antibodies (e.g., GN3E9, GC1C10, GC5D1,GC4A10, and/or GF2D6) may be immobilized on a solid support. Examples ofsuch solid supports include plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, acrylic resins and such aspolyacrylamide and latex beads. Techniques for coupling antibodies tosuch solid supports are well known in the art. (Weir et al., “Handbookof Experimental Immunology” 4th Ed., Blackwell Scientific Publications,Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34Academic Press, N.Y. (1974).)

The simplest method to bind the antigen to the antibody-support matrixis to collect the beads in a column and pass the antigen solution downthe column. The efficiency of this method depends on the contact timebetween the immobilized antibody and the antigen, which can be extendedby using low flow rates. The immobilized antibody captures the antigenas it flows past. Alternatively, an antigen can be contacted with theantibody-support matrix by mixing the antigen solution with the support(e.g., beads) and rotating or rocking the slurry, allowing maximumcontact between the antigen and the immobilized antibody. After thebinding reaction has been completed, the slurry is passed into a columnfor collection of the beads. The beads are washed using a suitablewashing buffer and then the pure or substantially pure antigen iseluted.

An antibody or polypeptide of interest can be conjugated to a solidsupport, such as a bead. In addition, a first solid support such as abead can also be conjugated, if desired, to a second solid support,which can be a second bead or other support, by any suitable means,including those disclosed herein for conjugation of a polypeptide to asupport. Accordingly, any of the conjugation methods and means disclosedherein with reference to conjugation of a polypeptide to a solid supportcan also be applied for conjugation of a first support to a secondsupport, where the first and second solid support can be the same ordifferent.

Appropriate linkers, which can be cross-linking agents, for use forconjugating a polypeptide to a solid support include a variety of agentsthat can react with a functional group present on a surface of thesupport, or with the polypeptide, or both. Reagents useful ascross-linking agents include homo-bi-functional and, in particular,hetero-bi-functional reagents. Useful bi-functional cross-linking agentsinclude, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA,N-SPDP, SMCC and 6-HYNIC. A cross-linking agent can be selected toprovide a selectively cleavable bond between a polypeptide and the solidsupport. For example, a photolabile cross-linker, such as3-amino-(2-nitrophenyl)propionic acid can be employed as a means forcleaving a polypeptide from a solid support. (Brown et al., Mol. Divers.4-12 (1995); Rothschild et al., Nucl. Acids Res. 24:351-66 (1996); andU.S. Pat. No. 5,643,722). Other cross-linking reagents are well-known inthe art. (See, e.g., Wong (1991), supra; and Hermanson (1996), supra).

An antibody or polypeptide can be immobilized on a solid support, suchas a bead, through a covalent amide bond formed between a carboxyl groupfunctionalized bead and the amino terminus of the polypeptide or,conversely, through a covalent amide bond formed between an amino groupfunctionalized bead and the carboxyl terminus of the polypeptide. Inaddition, a bi-functional trityl linker can be attached to the support,e.g, to the 4-nitrophenyl active ester on a resin, such as a Wang resin,through an amino group or a carboxyl group on the resin via an aminoresin. Using a bi-functional trityl approach, the solid support canrequire treatment with a volatile acid, such as formic acid ortrifluoracetic acid to ensure that the polypeptide is cleaved and can beremoved. In such a case, the polypeptide can be deposited as a beadlesspatch at the bottom of a well of a solid support or on the flat surfaceof a solid support. After addition of a matrix solution, the polypeptidecan be desorbed into a MS.

Hydrophobic trityl linkers can also be exploited as acid-labile linkersby using a volatile acid or an appropriate matrix solution, e.g., amatrix solution containing 3-HPA, to cleave an amino linked trityl groupfrom the polypeptide. Acid lability can also be changed. For example,trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can bechanged to the appropriate p-substituted, or more acid-labiletritylamine derivatives, of the polypeptide, i.e., trityl ether andtritylamine bonds can be made to the polypeptide. Accordingly, apolypeptide can be removed from a hydrophobic linker, e.g., bydisrupting the hydrophobic attraction or by cleaving tritylether ortritylamine bonds under acidic conditions, including, if desired, undertypical MS conditions, where a matrix, such as 3-HPA acts as an acid.

Orthogonally cleavable linkers can also be useful for binding a firstsolid support, e.g., a bead to a second solid support, or for binding apolypeptide of interest to a solid support. Using such linkers, a firstsolid support, e.g., a bead, can be selectively cleaved from a secondsolid support, without cleaving the polypeptide from the support; thepolypeptide then can be cleaved from the bead at a later time. Forexample, a disulfide linker, which can be cleaved using a reducingagent, such as DTT, can be employed to bind a bead to a second solidsupport, and an acid cleavable bi-functional trityl group could be usedto immobilize a polypeptide to the support. As desired, the linkage ofthe polypeptide to the solid support can be cleaved first, e.g., leavingthe linkage between the first and second support intact. Trityl linkerscan provide a covalent or hydrophobic conjugation and, regardless of thenature of the conjugation, the trityl group is readily cleaved in acidicconditions.

For example, a bead can be bound to a second support through a linkinggroup which can be selected to have a length and a chemical nature suchthat high density binding of the beads to the solid support, or highdensity binding of the polypeptides to the beads, is promoted. Such alinking group can have, e.g., “tree-like” structure, thereby providing amultiplicity of functional groups per attachment site on a solidsupport. Examples of such linking group; include polylysine,polyglutamic acid, penta-erythrole and tris-hydroxy-aminomethane.

Noncovalent Binding Association.

An antibody or polypeptide can be conjugated to a solid support, or afirst solid support can also be conjugated to a second solid support,through a noncovalent interaction. For example, a magnetic bead made ofa ferromagnetic material, which is capable of being magnetized, can beattracted to a magnetic solid support, and can be released from thesupport by removal of the magnetic field. Alternatively, the solidsupport can be provided with an ionic or hydrophobic moiety, which canallow the interaction of an ionic or hydrophobic moiety, respectively,with a polypeptide, e.g., a polypeptide containing an attached tritylgroup or with a second solid support having hydrophobic character.

A solid support can also be provided with a member of a specific bindingpair and, therefore, can be conjugated to a polypeptide or a secondsolid support containing a complementary binding moiety. For example, abead coated with avidin or with streptavidin can be bound to apolypeptide having a biotin moiety incorporated therein, or to a secondsolid support coated with biotin or derivative of biotin, such asimino-biotin.

It should be recognized that any of the binding members disclosed hereinor otherwise known in the art can be reversed. Thus, biotin, e.g., canbe incorporated into either a polypeptide or a solid support and,conversely, avidin or other biotin binding moiety would be incorporatedinto the support or the polypeptide, respectively. Other specificbinding pairs contemplated for use herein include, but are not limitedto, hormones and their receptors, enzyme, and their substrates, anucleotide sequence and its complementary sequence, an antibody and theantigen to which it interacts specifically, and other such pairs knowsto those skilled in the art.

A. Diagnostic Uses of Gelsolin Binding Agents

General.

The gelsolin binding compositions of the invention are useful indiagnostic methods. As such, the present invention provides methodsusing the binding agents of the invention in the diagnosis ofgelsolin-related medical conditions in a subject. Binding agents of theinvention may be selected such that they have any level of epitopebinding specificity and very high binding affinity to the gelsolinpolypeptide. In general, the higher the binding affinity of an bindingagent the more stringent wash conditions can be performed in animmunoassay to remove nonspecifically bound material without removingtarget polypeptide. Accordingly, gelsolin binding agents of theinvention may in diagnostic assays usually have binding affinities of atleast 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹² M⁻¹. Further, it is desirable thatgelsolin binding agents used as diagnostic reagents have a sufficientkinetic on-rate to reach equilibrium under standard conditions in atleast 12 h, preferably at least five (5) h and more preferably at leastone (1) hour.

Although gelsolin binding agents can be used as diagnostic reagents forany kind of sample, they are most useful as diagnostic reagents forhuman biological samples. Gelsolin binding agents can be used to detecta given gelsolin or gelsolin-like polypeptide in a variety of standardassay formats. Such formats include immunoprecipitation, Westernblotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow &Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications,New York (1988)); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752;3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752;3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074;3,984,533; 3,996,345; 4,034,074; and 4,098,876. Biological samples canbe obtained from any tissue or body fluid of a subject.

Immunometric or sandwich assays are a preferred format for thediagnostic methods of the present invention. See U.S. Pat. Nos.4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use onegelsolin binding agent, e.g., an anti-gelsolin antibody or a populationof anti-gelsolin antibodies immobilized to a solid phase, and anotheranti-gelsolin antibody or a population of anti-gelsolin antibodies insolution. Typically, the solution anti-gelsolin antibody or populationof anti-gelsolin antibodies is labeled. If an antibody population isused, the population can contain antibodies binding to different epitopespecificities within the target polypeptide. Accordingly, the samepopulation can be used for both solid phase and solution antibody. Ifanti-gelsolin monoclonal antibodies are used, first and second gelsolinmonoclonal antibodies having different binding specificities are usedfor the solid and solution phase. Solid phase (also referred to as“capture”) and solution (also referred to as “detection”) antibodies canbe contacted with target antigen in either order or simultaneously. Ifthe solid phase antibody is contacted first, the assay is referred to asbeing a forward assay. Conversely, if the solution antibody is contactedfirst, the assay is referred to as being a reverse assay. If the targetis contacted with both antibodies simultaneously, the assay is referredto as a simultaneous assay. After contacting the gelsolin polypeptidewith the anti-gelsolin antibody, a sample is incubated for a period thatusually varies from about 10 min to about 24 hr and is usually about 1hr. A wash step is then performed to remove components of the sample notspecifically bound to the anti-gelsolin antibody being used as adiagnostic reagent. When solid phase and solution antibodies are boundin separate steps, a wash can be performed after either or both bindingsteps. After washing, binding is quantified, typically by detecting alabel linked to the solid phase through binding of labeled solutionantibody. Usually for a given pair of antibodies or populations ofantibodies and given reaction conditions, a calibration curve isprepared from samples containing known concentrations of target antigen.Concentrations of the gelsolin polypeptide in samples being tested arethen read by interpolation from the calibration curve. Analyte can bemeasured either from the amount of labeled solution antibody bound atequilibrium or by kinetic measurements of bound labeled solutionantibody at a series of time points before equilibrium is reached. Theslope of such a curve is a measure of the concentration of the gelsolinpolypeptide in a sample

Suitable supports for use in the above methods include, e.g.,nitrocellulose membranes, nylon membranes, and derivatized nylonmembranes, and also particles, such as agarose, a dextran-based gel,dipsticks, particulates, microspheres, magnetic particles, test tubes,microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, PiscatawayN.J.), and the like. Immobilization can be by absorption or by covalentattachment. Optionally, anti-gelsolin antibodies can be joined to alinker molecule, such as biotin for attachment to a surface boundlinker, such as avidin.

The invention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with gelsolin polypeptide expression or activity. Such assayscan be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with a gelsolin polypeptide. Furthermore,the methods of the present invention can also be used to assess whetheran individual expresses a gelsolin polypeptide or a polymorphic form ofthe gelsolin polypeptide in instances where a gelsolin binding agent ofthe present invention has greater affinity for the gelsolin polypeptidefor its polymorphic form (or vice versa).

The binding of a gelsolin binding agent of the invention to a gelsolinpolypeptide or a gelsolin-like polypeptide, e.g., can be utilized toidentify a subject having or at risk of developing a disorder associatedwith altered gelsolin polypeptide or gelsolin-like polypeptide levels.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing the disease or disorder. Thus,the invention provides a method for identifying a disease or conditionassociated with an aberrant gelsolin polypeptide or gelsolin-likepolypeptide expression or activity in which a test sample is obtainedfrom a subject and the gelsolin or gelsolin-like polypeptide detected,wherein the presence of an alteration of gelsolin or gelsolin-likepolypeptide is diagnostic for a subject having or at risk of developinga disease or condition associated with an aberrant gelsolin polypeptideor gelsolin-like polypeptide expression or activity.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered a compound (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with an aberrant gelsolin polypeptide or gelsolin-likepolypeptide expression or activity. For example, such methods can beused to determine whether a subject can be effectively treated with acompound affecting gelsolin polypeptide levels (e.g., a chemotherapeuticagent). Thus, the invention provides methods for determining whether asubject can be effectively treated with a compound for a disorder orcondition associated with an aberrant gelsolin polypeptide orgelsolin-like polypeptide expression or activity in which a test sampleis obtained and the gelsolin polypeptide or the gelsolin-likepolypeptide is detected using the gelsolin binding agent (e.g., whereinthe presence or absence of the gelsolin polypeptide or the gelsolin-likepolypeptide is diagnostic for a subject that can be administered thecompound to treat a disorder associated with an aberrant gelsolinpolypeptide or gelsolin-like polypeptide expression or activity).

The level of the gelsolin polypeptide or the gelsolin-like polypeptidein a urine sample obtained from a subject is determined and comparedwith the level found in a urine sample obtained from an individual whois free of the disease or condition. An underabundance (oroverabundance) of the gelsolin polypeptide or gelsolin-like polypeptidein the sample obtained from the subject suspected of having the diseaseor condition affecting gelsolin levels compared with the sample obtainedfrom the healthy subject is indicative of the gelsolin polypeptide orgelsolin-like polypeptide-associated disease or condition in the subjectbeing tested. Further testing may be required to make a positivediagnosis. For example, an increased amount of gelsolin or gelsolin-likepolypeptide in the urine of a subject compared to a healthy control mayindicate the subject is suffering from a disease or condition causing anincreased utilization of plasma gelsolin.

There are a number of diseases in which the degree of underabundance (oroverabundance) of certain gelsolin polypeptide or gelsolin-likepolypeptide molecules known to be indicative of whether a subject withthe disease is likely to respond to a particular type of therapy ortreatment. Thus, the method of detecting a gelsolin polypeptide orgelsolin-like polypeptide in a sample can be used as a method ofprognosis, e.g., to evaluate the likelihood that the subject willrespond to the therapy or treatment. Examples of conditions in whichplasma gelsolin levels are decreased compared to control subjectsinclude, but are not limited to, septic shock, multiple organdysfunction syndrome, rheumatoid arthritis, trauma, stroke, heartinfarction, cancer, chemotherapy and radiation therapy, systemicautoimmune disease, and chronic hepatitis. Accordingly, urinegelsolin-like polypeptide levels would be increased in subjectssuffering from these conditions compared to healthy control subjects.

The methods described herein can be performed, e.g., by utilizingpre-packaged diagnostic kits comprising at least one probe reagent,e.g., gelsolin binding agent described herein, which can be convenientlyused, e.g., in clinical settings to diagnose subjects exhibitingsymptoms of a disease or illness involving a gelsolin polypeptide orgelsolin-like polypeptide. Furthermore, any cell type or tissue in whichgelsolin polypeptide or gelsolin-like polypeptide is expressed can beutilized in the prognostic assays described herein.

Correlating a Subject to a Standard Reference Population.

To deduce a correlation between clinical response to a treatment and aparticular level of plasma gelsolin, it is necessary to obtain data onthe clinical responses exhibited by a population of individuals whoreceived the treatment, i.e., a clinical population. This clinical datamay be obtained by retrospective analysis of the results of a clinicaltrial(s). Alternatively, the clinical data may be obtained by designingand carrying out one or more new clinical trials. The analysis ofclinical population data is useful to define a standard referencepopulation(s) which, in turn, are useful to classify subjects forclinical trial enrollment or for selection of therapeutic treatment. Ina preferred embodiment, the subjects included in the clinical populationhave been graded for the existence of the medical condition of interest.Grading of potential subjects can include, e.g., a standard physicalexam or one or more lab tests. Alternatively, grading of subjects caninclude use of a gene expression pattern. For example, plasma or urinegelsolin levels are useful as grading criteria where there is a strongcorrelation between expression pattern and susceptibility or severity toa disease or condition. In one embodiment, a subject is classified orassigned to a particular group or class based on similarity between themeasured levels of a one or more biomarkers in the subject and the levelof the one or more biomarkers observed in a standard referencepopulation.

In one embodiment of the invention, a therapeutic treatment of interestis administered to each subject in a trial population, and eachsubject's response to the treatment is measured using one or morepredetermined criteria. It is contemplated that in many cases, the trialpopulation will exhibit a range of responses, and that the investigatorwill choose the number of responder groups (e.g., low, medium, high)made up by the various responses. In addition, the expression level of abiomarker (e.g., urine gelsolin) is quantified, which may be done beforeand/or after administering the treatment. These results are thenanalyzed to determine if any observed variation in clinical responsebetween groups is statistically significant. Statistical analysismethods, which may be used, are described in L. D. Fisher & G. vanBelle,Biostatistics: A Methodology for the Health Sciences(Wiley-Interscience, New York (1993)).

The skilled artisan can construct a mathematical model that predictsclinical response as a function of expression level from the analysesdescribed above. The identification of an association between a clinicalresponse and an expression level for the biomarker may be the basis fordesigning a diagnostic method to determine those individuals who will orwill not respond to the treatment, or alternatively, will respond at alower level and thus may require more treatment, i.e., a greater dose ofa drug. The diagnostic method may take one of several forms: forexample, a ELISA or antibody-based test, a serological test, or aphysical exam measurement. The only requirement is that there be a goodcorrelation between the diagnostic test results and the underlyingcondition. In one embodiment, this diagnostic method uses an antibodyassay for serum or urine gelsolin as described above.

In one embodiment, the level of the biomarker molecule in a blood ortissue sample obtained from a first subject is determined and comparedwith the level found in a blood sample or a sample from the same tissuetype obtained from an second subject free of the biomarker-associateddisease. An overabundance (or underabundance) of the biomarker moleculein the sample obtained from the first subject suspected of having thebiomarker associated disease compared with the sample obtained from thehealthy (second) subject is indicative of the biomarker-associateddisease in the subject being tested. Further testing may be required tomake a positive diagnosis.

In one embodiment, the level of the biomarker molecule (e.g., gelsolin)in a blood, urine, or tissue sample obtained from a subject at a firsttime point is determined and compared with the level found in a urinesample obtained from the subject at a later time point. An overabundance(or underabundance) of the biomarker molecule in the sample obtainedfrom the subject at the first time point can be compared with the sampleobtained from the subject at the second time point wherein the increaseof the biomarker level between the first time point compared with thebiomarker level at the second time point is indicative of a subject whois in need of gelsolin replacement therapy. Further testing may berequired to make a positive diagnosis.

There are a number of diseases in which the degree of overexpression (orunderexpression) of certain biomarker molecules is known to beindicative of whether a subject with the disease is likely to respond toa particular type of therapy or treatment. Thus, the method of detectinga biomarker molecule in a sample can be used as a method of prognosis,e.g., to evaluate the likelihood that the subject will respond to thetherapy or treatment. Accordingly, in another embodiment, the level ofat least one biomarker molecules in a blood or urine sample obtainedfrom a first subject is determined and compared with the level of the atleast one biomarker molecules found in a blood or urine sample obtainedfrom a second subject, or standard reference population, responsive to acompound, e.g., a therapeutic compound of interest. Similarity in thelevel or pattern of expression of the at least one biomarker moleculesin a blood or urine sample obtained from a first subject compared withthe level of the at least one biomarker molecules found in a blood orurine sample obtained from the second subject, or standard referencepopulation, indicates that the first subject will be responsive to thecompound, e.g., therapeutic compound of interest. That is, the level ofthe relevant biomarker in a suitable tissue or biological sample fromthe subject is determined and compared with a suitable control, e.g.,the level in subjects with the same disease but who have respondedfavorably to the treatment. The degree to which the biomarker isoverexpressed (or underexpressed) in the sample compared with thecontrol may be predictive of likelihood that the subject will notrespond favorably to the treatment or therapy (e.g., toleratechemotherapy). The greater the overexpression (or underexpression)relative to the control, the less likely the subject will respond to thetreatment.

There are a number of diseases in which the degree of overexpression (orunderexpression) of certain biomarker molecules, i.e.,biomarker-associated disease or medical condition, is known to beindicative of whether a subject will develop a disease. Thus, the methodof detecting a biomarker in a sample can be used as a method ofpredicting whether a subject will develop a disease. The level of a oneor more biomarkers in a suitable urine or blood sample from a subject atrisk of developing the disease or condition is determined and comparedwith a suitable control, e.g., the level in subjects who are not at riskof developing the disease. The degree to which the one or morebiomarkers is overexpressed (or underexpressed) in the sample comparedwith the control may be predictive of likelihood that the subject willdevelop the disease. The greater the overexpression (or underexpression)relative to the control, the more likely the subject will developmentthe disease.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe reagent,e.g., anti-gelsolin polypeptide antibody described herein, which can beconveniently used, e.g., in clinical setting to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga biomarker of the invention. Furthermore, any cell type or tissue inwhich a biomarker of the invention is expressed can be utilized in theprognostic assays described herein.

Monitoring Clinical Efficacy.

In one embodiment, the present invention provides for monitoring theinfluence of agents (e.g., drugs, compounds, or small molecule) on theexpression or utilization of gelsolin or gelsolin-like polypeptides.Such assays can be applied in basic drug screening and in clinicaltrials. For example, the effectiveness of an agent to increase (ordecrease) gelsolin or gelsolin-like polypeptide levels can be monitoredin clinical trials of subjects exhibiting decreased expression ofgelsolin. An agent that affects the expression of gelsolin orgelsolin-like polypeptides can be identified by administering the agentand observing a response. In this way, the expression pattern of thegelsolin or gelsolin-like polypeptide can serve as a marker, indicativeof the physiological response of the subject to the agent. Accordingly,this response state may be determined before, and at various pointsduring, treatment of the individual with the agent.

Subject Classification.

Standard control levels of a gelsolin or gelsolin-like polypeptide aredetermined by measuring levels in different control groups. The controllevels (reference levels) are then compared with the measured level of agelsolin or gelsolin-like polypeptide in a given subject. The subjectcan be classified or assigned to a particular group based on how similarthe measured levels were compared to the reference levels for a givengroup.

As one of skill in the art will understand, there will be a certaindegree of uncertainty involved in making this determination. Therefore,the standard deviations of the control group levels can be used to makea probabilistic determination and the method of this invention areapplicable over a wide range of probability-based genotype groupdeterminations. Thus, for example, and not by way of limitation, in oneembodiment, if the measured level of the gelsolin polypeptide fallswithin 2.5 standard deviations of the mean of any of the control groups,then that individual may be assigned to that group. In anotherembodiment if the measured level of the gelsolin polypeptide fallswithin 2.0 standard deviations of the mean of any of the control groupsthen that individual may be assigned to that group. In still anotherembodiment, if the measured level of the gelsolin polypeptide fallswithin 1.5 standard deviations of the mean of any of the control groupsthen that individual may be assigned to that group. In yet anotherembodiment, if the measured level of the gelsolin polypeptide is 1.0 orless standard deviations of the mean of any of the control groups levelsthen that individual may be assigned to that group.

Thus, this process allows determination, with various degrees ofprobability, which group a specific subject should be placed in, andsuch assignment would then determine the risk category into which theindividual should be placed.

B. Kits

Also within the scope of the invention are kits comprising the gelsolinbinding agent compositions (e.g., monoclonal antibodies) of theinvention and instructions for use. The kits are useful for detectingthe presence of a gelsolin polypeptide or a gelsolin-like polypeptide ina biological sample e.g., any body fluid including, but not limited to,e.g., serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinalfluid, acitic fluid or blood and including biopsy samples of bodytissue. For example, the kit can comprise: one or more gelsolin bindingagents capable of binding a gelsolin polypeptide or a gelsolin-likepolypeptide in a biological sample (e.g., an antibody or antigen-bindingfragment thereof having the same antigen-binding specificity ofantibodies produced by a deposited cell line selected from the groupconsisting of: CGMCC Accession Nos: 2114, 2115, and 2116); means fordetermining the amount of the gelsolin polypeptide or gelsolin-likepolypeptide in the sample; and means for comparing the amount of thegelsolin polypeptide or the gelsolin-like polypeptide in the sample witha standard. One or more of the gelsolin binding agents may be labeled.The kit components, (e.g., reagents) can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect the gelsolin polypeptide or the gelsolin-like polypeptide.

For antibody-based kits, the kit can comprise, e.g., 1) a firstantibody, e.g., attached to a solid support, which binds to apolypeptide corresponding to a marker or the invention; and, optionally;2) a second, different antibody which binds to either the polypeptide orthe first antibody and is conjugated to a detectable label.

The kit can also comprise, e.g., a buffering agent, a preservative or aprotein-stabilizing agent. The kit can further comprise componentsnecessary for detecting the detectable-label, e.g., an enzyme or asubstrate. The kit can also contain a control sample or a series ofcontrol samples, which can be assayed and compared to the test sample.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package, alongwith instructions for interpreting the results of the assays performedusing the kit. The kits of the invention may contain a written producton or in the kit container. The written product describes how to use thereagents contained in the kit, e.g., to use the biomarkers of thepresent invention in determining a strategy for preventing or treating amedical condition in a subject. In several embodiments, the use of thereagents can be according to the methods of the invention.

C. Prophylactic and Therapeutic Use of Gelsolin Replacement Therapy

General. The gelsolin binding agents and methods of the presentinvention can be used in conjunction with gelsolin replacement therapy.Specifically, the invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with an aberrant gelsolinexpression or activity. The gelsolin binding agents and methods areused, for example, to ascertain the suitability of gelsolin replacementtherapy for a subject or monitor the efficacy of gelsolin replacementtherapy in a subject receiving such therapy. In one embodiment, atherapeutically effective amount of recombinant or purified, nativegelsolin compounds are administered so as to provide therapeuticbenefits against the secondary toxic effects of excessive extracellularactin. By “excessive” extracellular actin is meant an amount ofextracellular actin which exceeds the ability of the plasma proteins tobind and clear the actin from extracellular fluids without secondarytissue damage or toxic effects. By “secondary” tissue damage or toxiceffects is meant the tissue damage or toxic effects which occur tootherwise healthy tissues, organs, and the cells therein, due to thepresence of excessive extracellular actin in the plasma, usually as aresult of a “primary” tissue injury elsewhere in the body. While notwishing to be limited by theory, infusion of gelsolin, results in a)binding to actin monomers so as to prevent their condensation into actinfilaments, and/or b) cleavage of actin filaments to the monomeric state,and/or c) enhanced clearance of such actin complexed to actin-bindingproteins or fragments thereof from the circulation or extracellulartissue environment.

Optionally, the administration is made during the course of adjuncttherapy such as combined cycles of radiation, chemotherapeutictreatment, or administration of other cytoprotective or immunomodulatoryagent. As such the binding agents of the present invention and acompound useful in adjunct therapy may be administrated simultaneouslyand sequentially to a subject in need of administration thereof.

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant gelsolinexpression or activity, by administering to the subject gelsolin.Administration of a prophylactic gelsolin binding agent can occur priorto the manifestation of symptoms characteristic of the aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. In therapeutic applications, gelsolin is administeredto a subject suspected of, or already suffering from, reduced serumgelsolin levels. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose.

Determination of the Biological Effect of the gelsolin-BindingAgent-Based Therapeutic.

In various embodiments of the invention, suitable in vitro or in vivoassays are performed to determine the effect of gelsolin replacementtherapy and whether its administration is indicated for treatment of theaffected tissue in a subject.

Typically, an effective amount of gelsolin, sufficient for achieving atherapeutic or prophylactic effect, range from about 0.000001 mg perkilogram body weight per day to about 10,000 mg per kilogram body weightper day. Preferably, the dosage ranges are from about 0.0001 mg perkilogram body weight per day to about 100 mg per kilogram body weightper day. For administration of gelsolin, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg every week, everytwo weeks or every three weeks, of the host body weight. For exampledosages can be 1 mg/kg body weight or 10 mg/kg body weight every week,every two weeks or every three weeks or within the range of 1-10 mg/kgevery week, every two weeks or every three weeks. An exemplary treatmentregime entails administration once per every two days or once a week oronce every month. Gelsolin is usually administered on multipleoccasions. Intervals between single dosages can be daily, weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody in the subject. In some methods,dosage is adjusted to achieve a serum gelsolin concentration in thesubject of from about 75 μg/mL to about 125 μg/mL, 100 μg/mL to about150 μg/mL, from about 125 μg/mL to about 175 μg/mL, or from about 150μg/mL to about 200 μg/mL. Alternatively, gelsolin can be administered asa sustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the gelsolin binding agent in the subject. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some subjects continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the subject shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Toxicity.

Preferably, an effective amount (e.g., dose) of gelsolin describedherein will provide therapeutic benefit without causing substantialtoxicity to the subject. Toxicity of the gelsolin described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD₅₀ (the dose lethal to50% of the population) or the LD₁₀₀ (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is thetherapeutic index. The data obtained from these cell culture assays andanimal studies can be used in formulating a dosage range that is nottoxic for use in human. The dosage of the gelsolin described herein liespreferably within a range of circulating concentrations that include theeffective dose with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thesubject's condition. See, e.g., Fingl et al., In: The PharmacologicalBasis of Therapeutics Ch. 1 (1975).

Formulations of Pharmaceutical Compositions.

According to the methods of the present invention, the gelsolin can beincorporated into pharmaceutical compositions suitable foradministration. The pharmaceutical compositions generally compriserecombinant or substantially purified native gelsolin and apharmaceutically-acceptable carrier in a form suitable foradministration to a subject. Pharmaceutically-acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations ofpharmaceutical compositions for administering the protein compositions.(See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 18^(th) ed. (1990).) The pharmaceutical compositions aregenerally formulated as sterile, substantially isotonic and in fullcompliance with all Good Manufacturing Practice (GMP) regulations of theU.S. Food and Drug Administration.

The terms “pharmaceutically-acceptable,” “physiologically-tolerable,”and grammatical variations thereof, as they refer to compositions,carriers, diluents and reagents, are used interchangeably and representthat the materials are capable of administration to or upon a subjectwithout the production of undesirable physiological effects to a degreethat would prohibit administration of the composition. For example,“pharmaceutically-acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. “Pharmaceutically-acceptable salts andesters” means salts and esters that are pharmaceutically-acceptable andhave the desired pharmacological properties. Such salts include saltsthat can be formed where acidic protons present in the gelsolin arecapable of reacting with inorganic or organic bases. Suitable inorganicsalts include those formed with the alkali metals, e.g., sodium andpotassium, magnesium, calcium, and aluminum. Suitable organic saltsinclude those formed with organic bases such as the amine bases, e.g.,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Such salts also include acid additionsalts formed with inorganic acids (e.g., hydrochloric and hydrobromicacids) and organic acids (e.g., acetic acid, citric acid, maleic acid,and the alkane- and arene-sulfonic acids such as methanesulfonic acidand benzenesulfonic acid). Pharmaceutically-acceptable esters includeesters formed from carboxy, sulfonyloxy, and phosphonoxy groups presentin the gelsolin, e.g., C₁₋₆ alkyl esters. When there are two acidicgroups present, a pharmaceutically-acceptable salt or ester can be amono-acid-mono-salt or ester or a di-salt or ester; and similarly wherethere are more than two acidic groups present, some or all of suchgroups can be salified or esterified. The gelsolin polypeptide can bepresent in unsalified or unesterified form, or in salified and/oresterified form, and the naming of such gelsolin polypeptide is intendedto include both the original (unsalified and unesterified) compound andits pharmaceutically-acceptable salts and esters. Also, certain gelsolinpolypeptides can be present in more than one stereoisomeric form, andthe naming of such gelsolin polypeptide is intended to include allsingle stereoisomers and all mixtures (whether racemic or otherwise) ofsuch stereoisomers. A person of ordinary skill in the art, would have nodifficulty determining the appropriate timing, sequence and dosages ofadministration for particular drugs and compositions of the presentinvention.

Examples of such carriers or diluents include, but are not limited to,water, saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and compounds for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or compound is incompatible with the gelsolin binding agent, usethereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. The gelsolincompositions can be administered by parenteral, topical, intravenous,oral, subcutaneous, intraarterial, intradermal, transdermal, rectal,intracranial, intraperitoneal, intranasal; intramuscular route or asinhalants. The gelsolin can optionally be administered in combinationwith other agents that are at least partly effective in treating variousdiseases including various actin- or microfilament-related diseases.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates, and compounds for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, e.g., water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, e.g., by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalcompounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic compounds, e.g., sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition a compound which delays absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thegelsolin in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the agent into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The agents can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, thepolypeptide can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding compounds, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating compound such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningcompound such as sucrose or saccharin; or a flavoring compound such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the gelsolin is delivered in the formof an aerosol spray from pressured container or dispenser which containsa suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, e.g., fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the gelsolin is formulated into ointments, salves, gels, or creams asgenerally known in the art.

The gelsolin can also be prepared as pharmaceutical compositions in theform of suppositories (e.g., with conventional suppository bases such ascocoa butter and other glycerides) or retention enemas for rectaldelivery.

In one embodiment, the gelsolin is prepared with carriers that willprotect the gelsolin against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically-acceptable carriers. These can beprepared according to methods known to those skilled in the art, e.g.,as described in U.S. Pat. No. 4,522,811.

Preparation of Recombinant Gelsolin.

Most of the discussion below pertains to production of gelsolin byculturing cells transformed with a vector containing a nucleic acidencoding gelsolin and recovering the polypeptide from the cell culture.It is further envisioned that the gelsolin may be produced by purifyingnative gelsolin from plasma using affinity purification (describedabove). The proteins and DNA sequences encoding the proteins may beproduced using standard methods. Purification can also be accomplishedusing standard procedures such as isolating proteins usingchromatography, using antibodies directed against the polypeptides, orby producing the polypeptides in a form in which they are fused to amoiety (or tag) that aids in purification and which can then be cleaved.

A polynucleotide encoding a gelsolin or gelsolin-like polypeptide (forexample, the polypeptide of SEQ ID NO.: 1) may be inserted into any ofthe many commercially available expression vectors using reagents andtechniques that are well known in the art. In preparing the recombinantexpression constructs, the various polynucleotides of the presentinvention may be inserted or substituted into a bacterialplasmid-vector. Any convenient plasmid may be employed, which will becharacterized by having a bacterial replication system, a marker whichallows for selection in a bacterium and generally one or more unique,conveniently located cloning sites. Numerous plasmids, also referred toas vectors, are available for transformation. Suitable vectors include,but are not limited to, the following: viral vectors, such as lambdavector system gt11, Charon 4, and plasmid vectors such as pBR322,pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,pKC37, pKC101, SV 40, pBluescript II SK+/−. or KS+/−(Stratagene, LaJolla, Calif.), and any derivatives thereof. Also suitable are yeastexpression vectors, which may be highly useful for cloning andexpression. Exemplary yeast plasmids include, without limitation, pPICZ,and pFLD. (Invitrogen, Carlsbad, Calif.). The selection of a vector willdepend on the preferred transformation technique and target host cells.

The nucleic acid molecule encoding gelsolin is inserted into a vector inthe 5′ to 3′ direction, such that the open reading frame is properlyoriented for the expression of the encoded protein under the control ofa promoter of choice. In this way, the gelsolin structural gene is saidto be “operably linked” to the promoter. Single or multiple nucleicacids may be inserted into an appropriate vector in this way, each underthe control of suitable promoters, to prepare a nucleic acid constructof the present invention.

Certain regulatory sequences may also be incorporated into theexpression constructs of the present invention. These includenon-transcribed regions of the vector, which interact with host cellularproteins to carry out transcription and translation. Such elements mayvary in their strength and specificity. Depending on the vector systemand host utilized, any number of suitable transcription and/ortranslation elements, including constitutive, inducible, and repressiblepromoters, as well as minimal 5′ promoter elements may be used.

A constitutive promoter is a promoter that directs constant expressionof a gene in a cell. Examples of some constitutive promoters that arewidely used for inducing expression of heterologous polynucleotidesinclude the ADH1 promoter for expression in yeast, those derived fromany of the several actin genes, which are known to be expressed in mosteukaryotic cell types, and the ubiquitin promoter, which is the promoterof a gene product known to accumulate in many cell types. Examples ofconstitutive promoters for use in mammalian cells include the RSVpromoter derived from Rous sarcoma virus, the CMV promoter derived fromcytomegalovirus, β-actin and other actin promoters, and the EF1αpromoter.

Also suitable as a promoter in the plasmids of the present invention isa promoter that allows for external control over the regulation of geneexpression. One way to regulate the amount and the timing of geneexpression is to use an inducible promoter. Unlike a constitutivepromoter, an inducible promoter is not always optimally active. Aninducible promoter is capable of directly or indirectly activatingtranscription of one or more DNA sequences or genes in response to aninducing agent (or inducer). Some inducible promoters are activated byphysical means, such as the heat shock promoter (HSP), which isactivated at certain temperatures. Other promoters are activated by achemical means, for example, IPTG. Other examples of inducible promotersinclude the metallothionine promoter, which is activated by heavy metalions, and hormone-responsive promoters, which are activated by treatmentof certain hormones. In the absence of an inducer, the nucleic acidsequences or genes under the control of the inducible promoter will notbe transcribed or will only be minimally transcribed. Promoters of thenucleic acid construct of the present invention may be either homologous(derived from the same species as the host cell) or heterologous(derived from a different species than the host cell).

Once the nucleic acid construct of the present invention has beenprepared, it may be incorporated into a host cell. This is carried outby transforming or transfecting a host or cell with a plasmid constructof the present invention, using standard procedures known in the art,such as described by Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor: Cold Spring Harbor LaboratoryPress, New York (2001). Suitable hosts and cells for the presentinvention include, without limitation, bacterial cells, virus, yeastcells, insect cells, plant cells, and mammalian cells, including humancells, as well as any other cell system that is suitable for producing arecombinant protein. Exemplary bacterial cells include, withoutlimitation, E. coli and Mycobacterium sp. Exemplary yeast hosts includewithout limitation, Pischia pastoris, Saccharomyces cerevisiae, andSchizosaccharomyces pombe. Methods of transformation or transfection mayresult in transient or stable expression of the genes of interestcontained in the plasmids. After transformation, the transformed hostcells can be selected and expanded in suitable culture. Transformedcells are first identified using a selection marker simultaneouslyintroduced into the host cells along with the nucleic acid construct ofthe present invention. Suitable markers include markers encoding forantibiotic resistance, such as resistance to kanamycin, gentamycin,ampicillin, hygromycin, streptomycin, spectinomycin, tetracycline,chloramphenicol, and the like. Any known antibiotic-resistance markercan be used to transform and select transformed host cells in accordancewith the present invention. Cells or tissues are grown on a selectionmedium containing an antibiotic, whereby generally only thosetransformants expressing the antibiotic resistance marker continue togrow. Additionally, or in the alternative, reporter genes, including,but not limited to, β-galactosidase, β-glucuronidase, luciferase, greenfluorescent protein (GFP) or enhanced green fluorescent protein (EGFP),may be used for selection of transformed cells. The selection markeremployed will depend on the target species.

To obtain the gelsolin protein, expression is induced if the codingsequences is under the control of an inducible promoter. To isolate theprotein, the host cell carrying an expression vector is propagated,homogenized, and the homogenate is centrifuged to remove bacterialdebris. The supernatant is then subjected to sequential ammonium sulfateprecipitation. The fraction containing the protein of the presentinvention is subjected to gel filtration in an appropriately sizeddextran or polyacrylamide column to separate the proteins. If necessary,the protein fraction may be further purified by HPLC. Alternativemethods of protein purification may be used as suitable. See J. E.Coligan et al., eds., Current Protocols in Protein Science (John Wiley &Sons (2003)). Upon obtaining the substantially purified recombinantprotein, the protein may be administered to a subject as describedherein. [Alternatively, recombinant gelsolin may be purified usingstandard anion exchange chromatography. Oberley, R. E. et al., Am JPhysiol Lung Cell Mol Physiol 287:L296-306 (2004).]

The following EXAMPLES are presented in order to more fully illustratesome embodiments of the invention. These EXAMPLES should in no way beconstrued as limiting the scope of the invention, as defined by theappended claims.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1 Purification of GN3E9, GC1C10, and GF2D6 Monoclonal Antibodies

The monoclonal anti-gelsolin antibodies GN3E9, GC1C10, and GF2D6 wereprepared as described in PCT Application No. PCT/CN2007/002467. Theisotype of selected anti-gelsolin antibodies was determined by goatanti-murine isotype specific antibodies (SouthernBiotech, Birmingham,Ala.). The isotype of GN3E9 was determined as murine IgG2b kappa, andthe isotype of GC and GF2D6 were determined as murine IgG1 kappa.

GN3E9 was purified by affinity chromatography using Sepharose GL-4Baffinity purification medium (Pharmacia, Uppsala, Sweden). The GC1C10and GF2D6 antibodies were purified by affinity chromatograpy usingProtein G-Sepharose CL-4B affinity purification medium (Pharmacia,Uppsala, Sweden). The culture supernatants were applied to the columnwith a flow rate of 2 ml per min. After the culture supernatant waspassed through the column, it was washed with 50 ml PBS. The protein waseluted with elution buffer (0.1 M glycine (pH 2.4), 0.15 M NaCl). Theoptical density of each eluted fraction (1 ml) was measured at OD280 nm.The fractions with OD280>0.1 units were collected. After addition of 100μl of neutralization buffer (1M Tris-HCl pH 8.5) to the fraction, theeluates were placed separately in dialysis tubing, and the eluatesdialyzed against 1 L of PBS (pH 7.5) at 4° C. The dialysis buffer waschanged twice. Each affinity purified antibody was concentrated to 1mg/ml, sterilized and stored at 4° C. until use.

Example 2 Immunoprecipitation of Human Urine Gelsolin with GelsolinBinding Agents of the Invention

Immunoprecipitation of Urine Gelsolin Polypeptides.

To determine the ability of gelsolin binding agents of the invention toimmunoprecipitate the urine form(s) of gelsolin, GN3E9 and GC antibodieswere conjugated to CNBr-activated Sepharose 4B (Amersham PharmaciaBiotech (Piscataway, N.J.), at a concentration of 2 mg/ml beads. Theconjugation procedure was performed according to the manufacturer'sinstructions. Briefly, the pre-activated beads (660 mg; equal toapproximately 2 mL final bead volume) were suspended in 15 volumes of 1mM HCl and allowed to swell for 30 min. The beads were then washed with15 gel volumes of cold (4° C.) 1 mM HCl followed by a wash with 15volumes of coupling buffer (0.1M NaHCO₃ pH 8.3 containing 0.5 M NaCl) toyield beads that are referred to as “washed gel”. Each anti-gelsolinantibody (GN3E9, GC1C10, and GF2D6) was diluted in coupling buffer to0.5 to 1.0 mg/ml and the pH was adjusted to pH 8.3. The washed gel wasadded to each anti-gelsolin antibody solution and the mixtures wereincubated overnight at 4° C. to yield “coupled gels”. The coupled gelswere resuspended in 15 volumes of 1 M ethanolamine for 2-4 h at roomtemperature to block unused activated chemical conjugation sites on theactivated beads. The blocked gels were then washed 8 times in 15 volumeswith alternating 50 mM Tris, 1 M NaCl pH 8.0 and 50 mM glycine, 1 M NaClpH 3.5 buffers followed by a final wash with 10 gel volumes of PBS toremove any unbound material.

Next, one milliliter (1 mL) human urine samples, which had beencentrifuged briefly to remove sediment, were incubated with 10 μlanti-gelsolin antibody-conjugated beads (i.e., GN3E9 or GC conjugatedbeads) at room temperature for 2 h. Unbound material was rinsed from theanti-gelsolin antibody-conjugated beads or blank beads by pelleting thebead by centrifugation (14,000 rpm, 3 min), removing the supernatant andthen washing the pelleted beads by resuspension in PBS. Following five(5) wash cycles, material bound to the anti-gelsolin antibody-conjugatedbeads was removed under denaturing conditions by adding 40 μl SDS-PAGEloading buffer (SDS-PAGE loading buffer prepared by mixing 3× stock: 1MTris-Cl pH 6.8 2.4 ml; 20% SDS 3 ml; Glycerol (100%) 3 ml;B-mercaptoethanol 1.6 ml; Bromophenol blue 0.006 g, 10 ml) to thepelleted beads and boiling the sample for 5 min. The immunoprecipitatedproteins were fractionated on a 10% SDS-PAGE and visualized stained withCoomassie blue stain using standard techniques. After destain, the gelwas scanned using an HP photographic scanner. The results are shown inFIG. 1. The top panel shows the results of immunopreciptiation byN-terminal specific anti-gelsolin antibody GN3E9. The bottom panel showsthe results of immunopreciptiation by C-terminal specific anti-gelsolinantibody GC1C10. The lanes of the SDS-PAGE shown in FIG. 1 are asfollows: Lanes 1-3: healthy control patients; lanes 4-8: severe strokepatients from the ICU.

The C-terminal specific anti-gelsolin antibodies of the invention(GC1C10 and GF2D6) were able to immunoprecipitate two ˜50 kDapolypeptides from ICU patients, but not from healthy control patients.The N-terminal specific anti-gelsolin antibody GN3E9 was unable toimmunoprecipitate these fragments from either control or ICU patients.As such, the gelsolin binding agents of the invention tested in thepresent studies were able to identify ˜50 kDa immunoreactivepolypeptides from human urine samples. The identity of these ˜50 kDapolypeptides was confirmed to be C-terminal gelsolin fragments by massspectrometry analysis. (See Example 2.)

The ability of the select gelsolin binding agents of the inventiontested to immunoprecipitate gelsolin-like polypeptide is advantageousfor use of these binding agents in methods of the present invention.Specifically, gelsolin binding agents of the invention which canprecipitate immunoreactive gelsolin polypeptides can be employed todetect gelsolin-like fragments in urine samples.

Western Blot Analysis.

A western blot analysis technique was used to assess theimmunopreciptiation of urine gelsolin fragments from the biologicalsamples. That is, to further determine the identity of the urinegelsolin fragments, western blot analysis of immunoprecipitated humanurine proteins with anti-gelsolin antibodies was performed. The urinesamples of three normal human subjects and three severe stroke (ICU)subjects were immunoprecipitated with the C-terminal-specificanti-gelsolin antibodies GN3E9 or GC1C10 as described above.Immunoprecipitated proteins were fractionated by 10% SDS-PAGE andwestern blotted onto nitrocellulose membrane using standard techniques.After blocking the electroblotted nitrocellulose membrane (blot) using5% (w/v) non-fat milk at room temperature for 1 h, the blots were probedwith the purified anti-gelsolin antibodies GN3E9 or GC1C10 coupled toHRP (1 μg/ml) at room temperature for 2 h. Unbound anti-gelsolinantibody was rinsed from the blots by washing with PBS containing 0.02%Tween 20 at room temperature for 10 min with shaking. The complexes werevisualized using HRP-mediated chemiluminescence. Specifically, the blotswere incubated with LumiGLO® Peroxidase Chemiluminescent Substrate (KPL,Gaithersburg, Md.) for 3 min, and exposed to X-ray films. The resultsare shown in FIG. 2. Except for the sample in lane 6, the N-terminalspecific antibody GN3E9 did not detect any fragments other thanfull-length gelsolin in urine samples. As shown in FIG. 2B, asignificant amount of immunoreactive peptides corresponding to fragmentsof human gelsolin were identified in the samples obtained from ICUsubjects (lanes 4-8), but not the control subjects (lanes 1-3). Thus,the gelsolin binding agent of the invention can detect immunoreactivegelsolin-like polypeptides in urine samples in a Western Blot assay.

Example 3 Characterization of the Immunoreactive Polypeptide Bound byGelsolin Binding Agents

To confirm that the proteins immunoprecipitated by anti-gelsolinantibodies are fragments of human gelsolin, protein bands were cut fromSDS-PAGE and the contents subjected to analysis by mass spectroscopy atthe National Center of Biomedical Analysis (Beijing, China) usingstandard techniques (see Lewis et al., Identification of Viral Mutantsby Mass Spectrometry, Proc Nat Acad Sci USA 95:8596-8601 (1998)). Urineproteins were immunoprecipitated as described in Example 1 usingC-terminal specific anti-gelsolin antibody GF2D6. The individual proteinbands that were analyzed are shown in FIG. 1. The fragments F1 and F2were cut from the gel and subjected to analysis by mass spectroscopy.The peptides identified by mass spectrometry from fragments F1 and F2are indicated by the underlined regions of Tables 5 and 6, respectively.

TABLE 5 Peptide Fingerprint of Fragment F1 (SEQ ID NO.: 6) 401QTDGLGLSYL SSHIANVERV PFDAATLHTS TAMAAQHGMD DDGTGQKQIW 451RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ 501DEVAASAILT AQLDEELGGT PVQSRVVQGK EPAHLMSLFG GKPMIIYKGG 551TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPS 601AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA 651YRTSPRLKDK KMDAHPPRLF ACSNKIGRFV IEEVPGELMQ EDLATDDVML 701LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITVVKQG 751FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL AA

TABLE 6 Peptide Fingerprint of Fragment F2 (SEQ ID NO.: 7) 401QTDGLGLSYL SSHIANVERV PFDAATLHTS TAMAAQHGMD DDGTGQKQIW 451RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ 501DEVAASAILT AQLDEELGGT PVQSRVVQGK EPAHLMSLFG GKPMIIYKGG 551TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPS 601AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA 651YRTSPRLKDK KMDAHPPRLF ACSNKIGRFV IEEVPGELMQ EDLATDDVML 701LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITVVKQG 751FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL AA

A proteomics database (Swiss-Prot/TrEMBL) search indicated that thepolypeptides were C-terminal fragments of human gelsolin. N-terminalsequence of the F1 fragment was obtained and showed that the F1 fragmentcorresponds to amino acids 410-782 of SEQ ID NO: 1. Accordingly, the ˜50kDa polypeptides which were immunoprecipitated by the anti-gelsolinantibodies of the invention were confirmed as C-terminal fragments humangelsolin.

Example 4 Antibody Pairs Specific for C-Terminal Fragments of HumanUrine Gelsolin

Immunoprecipitation of Urine Gelsolin C-Terminal Fragments. To determinethe ability of gelsolin binding agents of the invention toimmunoprecipitate fragment(s) of urine gelsolin, the GC5D1 antibody wasconjugated to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech(Piscataway, N.J.), at a concentration of 2 mg/ml beads. The conjugationprocedure was performed according to the manufacturer's instructions.Briefly, the pre-activated beads (660 mg; equal to approximately 2 mLfinal bead volume) were suspended in 15 volumes of 1 mM HCl and allowedto swell for 30 min. The beads were then washed with 15 gel volumes ofcold (4° C.) 1 mM HCl followed by a wash with 15 volumes of couplingbuffer (0.1M NaHCO₃ pH 8.3 containing 0.5 M NaCl) to yield beads thatare referred to as “washed gel”. The GC5D1 antibody was diluted incoupling buffer to 0.5 to 1.0 mg/ml and the pH was adjusted to pH 8.3.The washed gel was added to each anti-gelsolin antibody solution and themixtures were incubated overnight at 4° C. to yield “coupled gels”. Thecoupled gels were resuspended in 15 volumes of 1 M ethanolamine for 2-4h at room temperature to block unused activated chemical conjugationsites on the activated beads. The blocked gels were then washed 8 timesin 15 volumes with alternating 50 mM Tris, 1 M NaCl pH 8.0 and 50 mMglycine, 1 M NaCl pH 3.5 buffers followed by a final wash with 10 gelvolumes of PBS to remove any unbound material.

Next, one milliliter (1 mL) human urine samples from controls and severestroke patients, which had been centrifuged briefly to remove sediment,was incubated with 10 μl GC5D1 antibody-conjugated beads at roomtemperature for 2 h. Unbound material was rinsed from the anti-gelsolinantibody-conjugated beads by pelleting the bead by centrifugation(14,000 rpm, 3 min), removing the supernatant and then washing thepelleted beads by resuspension in PBS. Following five (5) wash cycles,materials bound to the GC5D 1 antibody-conjugated beads was removedunder denaturing conditions by adding 40 μl SDS-PAGE loading buffer(SDS-PAGE loading buffer prepared by mixing 3× stock: 1M Tris-Cl pH 6.82.4 ml; 20% SDS 3 ml; Glycerol (100%) 3 ml; B-mercaptoethanol 1.6 ml;Bromophenol blue 0.006 g, 10 ml) to the pelleted beads and boiling thesample for 5 min. The immunoprecipitated proteins were fractionated on a10% SDS-PAGE and western blotted onto nitrocellulose membrane usingstandard techniques. After blocking the electroblotted nitrocellulosemembrane (blot) using 5% (w/v) non-fat milk at room temperature for 1 h,the blots were probed with GF2D6 coupled to HRP (1 μg/ml) at roomtemperature for 2 h. Unbound anti-gelsolin antibody was rinsed from theblots by washing with PBS containing 0.02% Tween 20 at room temperaturefor 10 min with shaking. The complexes were visualized usingHRP-mediated chemiluminescence. Specifically, the blots were incubatedwith LumiGLO® Peroxidase Chemiluminescent Substrate (KPL, Gaithersburg,Md.) for 3 min, and exposed to X-ray films. The results are shown inFIG. 3.

The C-terminal specific anti-gelsolin antibodies of the invention(GC5D1, GF2D6) were able to immunoprecipitated and detect two ˜50 kDapolypeptides from ICU patients, but not from healthy control patients.The full-length gelsolin polypeptide was not detected using thecombination of these two antibodies. As such, the gelsolin bindingagents of the invention tested in the present studies were able toidentify ˜50 kDa immunoreactive polypeptides from human urine samples.

The ability of the select gelsolin binding agents of the inventiontested to immunoprecipitate is advantageous for use of these bindingagents in methods of the present invention. Specifically, gelsolinbinding agents of the invention which can precipitate immunoreactivegelsolin polypeptides can be employed to detect gelsolin-likepolypeptides in urine samples.

Example 5 Quantitaive Measurement of Urine Gelsolin Fragments UsingGelsolin Binding Agents of the Invention Anti-gelsolin antibodies weretested to determine their ability to serve as a capture or detectionantibody for the detection of urine gelsolin fragments. Gelsolin ELISAwas carried out as follows. An ELISA plate (96 well; BD biosciences CA)was coated with 10 μg/ml of the capture antibody (GC5D 1) at 4° C.overnight. Unbound capture antibody was rinsed from the wells by washingthe plate three times with PBS. Non-specific binding sites were thenblocked by incubating the wells with 3% (w/v) BSA in PBS (1 h, roomtemperature). Blocking solution was removed from the wells and the platewas air dried prior to vacuum sealing and storage at 4° C. prior to use.

Varying concentrations (0.01 to 1,000 ng/mL) of recombinant gelsolinimmunogens were added to the ELISA plates. The immunogens were eitherfull-length gelsolin (FL), an N-terminal (NT) fragment comprising aminoacids 13 to 440 of SEQ ID NO: 1, or a C-terminal fragment (CT)comprising amino acids 440 to 782 of SEQ ID NO: 1. Unbound gelsolinimmunogen was rinsed from the wells by washing three times with PBS.

Bound gelsolin immunogen was detected by incubating wells (30 min; 37°C.) with HRP-conjugated 2D6 antibody (1:10,000). Unbound HRP-conjugated2D6 antibody was removed by rinsing the wells three times with PBS (5min each). The antibody complexes were measured using SureBlue TMB1-Component Microwell Peroxidase Substrate (KPL, Gaithersburg, Md.).Specifically, SureBlue TMB 1-Component Microwell Peroxidase Substrate(KPL, Gaithersburg, Md.) was added to the wells and the plate wasincubated for 10 min to allow HRP-mediated conversion of the substrate.The enzymatic reaction was stopped with the addition of 100 μl 2N H₂SO₄.The optical density of the sample wells was then measured at 450 nm/650nm using an ELISA plate reader.

A summary of the results of studies performed to determine the bindingcharacteristics of select gelsolin binding agents as detailed below areshown in FIG. 4. The C-terminal antibody pair showed a dose-dependentresponse to the C-terminal gelsolin immunogen, but the N-terminal orfull-length immunogens were not significantly detected over the range ofconcentrations tested. As such, select gelsolin binding agents have theability to specifically detect urine gelsolin fragments in aquantitative manner. Therefore, these gelsolin binding agents can beemployed to detect gelsolin-like polypeptides in urine samples in anELISA format.

Example 6 Detection of Urine Gelsolin Fragments in Clinical Samples

To test the ability of the gelsolin ELISA assay to quantitate urinegelsolin fragments in a clinical setting, samples from normal patientsand trauma patients were obtained and analyzed using an ELISA assay asdescribed in Example 5. Samples were obtained from subjects sufferingfrom various kinds of trauma or medical conditions. These includedcritical care (ICU) patients and cancer patients (i.e. patients withnewly diagnosed cancer prior to any major treatment such as surgery,chemotherapy, or radiation therapy). Further samples were obtained frompatients having major surgery, where major surgery is defined as anysurgical procedure that involves anesthesia or respiratory assistance.The criteria for patient recruitment was previously described. (See Wanget al., Eur J Clin Pharmacol 62:927-31 (2006).) Sepsis patents includedthose patients with sepsis associated with new organ dysfunction,hypotension, or hypoperfusion. The criteria for patient recruitment waspreviously described. (See Chen et al., Genes Immun 8:439-43 (2007).)Finally, samples were obtained from patients having nephritis, which isan inflammation of the kidney.

The results are shown in FIG. 5. Normal patients had a very low level ofurine gelsolin fragments, while kidney failure, critical care, cancer,post-surgery, sepsis, and nephritis patients exhibited elevated levelsof urine gelsolin, as measured by the assay (FIGS. 5A and 5B). Theseresults demonstrate the ability of the gelsolin binding agents of thepresent invention to quantify urine gelsolin fragments in a clinicalsetting. Further, the studies demonstrate that gelsolin-likepolypeptides are a biomarker of human kidney failure, stroke, sepsis,cancer, trauma, and nephritis. Moreover, the gelsolin binding agents ofthe invention may be used to measure the subsequent response of apatient to gelsolin replacement therapy by measuring serum gelsolinlevel after treatment of the patient.

Example 7 Inverse Correlation Between Full-Length Plasma Gelsolin andUrine Gelsolin Fragments

In this example, the relationship between full-length plasma gelsolinlevels and urine gelsolin fragments was investigated usingpatient-matched samples from either normal individuals or critical carepatients. The level of urine gelsolin fragments was measured asdescribed in FIG. 5.

Gelsolin ELISA for plasma samples was carried out as follows. An ELISAplate (96 well; BD biosciences CA) was coated with 10 μg/ml of thecapture antibody GN3E9 at 4° C. overnight. Unbound capture antibody wasrinsed from the wells by washing the plate three times with PBS.Non-specific binding sites were then blocked by incubating the wellswith 3% (w/v) BSA in PBS (1 h, room temperature). Blocking solution wasremoved from the wells and the plate was air dried prior to vacuumsealing and storage at 4° C. prior to use. Fifty (50) μl of human plasmawas first added to appropriate wells of the gelsolin ELISA plate treatedwith capture antibody which had been equilibrated to room temperature.Immediately following the addition of the samples to the plate, 50 μl ofHRP-conjugated detection antibody GC1C10 (˜0.1 μg/ml in blocking buffer)was added to appropriate wells and the plate was incubated for 20 min at37° C. Unbound material was rinsed from the wells by washing the platethree times with PBS. Captured plasma gelsolin: HRP-conjugated antibodycomplexes were measured by adding 100 μl ECL substrate buffer (KPL,Inc., Gaithersburg, Md.) was added. After incubation at 37° C. for 3min, the optical density of each well was measured at 450 nm/650 nm inan ELISA plate reader.

The results are shown in FIG. 6 and indicate that critical care patientswith elevated levels of urine gelsolin fragments also had depletedlevels of plasma gelsolin. Likewise, healthy patients with low levels ofurine gelsolin fragments had higher levels of plasma gelsolin. As such,there is an inverse correlation between plasma gelsolin levels and thelevels of urine gelsolin fragments.

Example 7 Correlation of Levels of Urine Gelsolin Fragments to ClinicalOutcome

To test the ability of the gelsolin ELISA assay to quantitate urinegelsolin fragments in a clinical setting, samples from critical carepatients are obtained and analyzed as described above. Clinical outcomeor disease stage for each patient is assessed. A mathematical model isconstructed that predicts clinical response or outcome as a function ofurine gelsolin fragment level. The identification of an associationbetween clinical response and a level for the urine gelsolin fragmentsis a basis for designing a diagnostic method to determine those subjectswho will or will not respond to the treatment, or alternatively, willrespond at a lower level and thus require more treatment, i.e., agreater dose of a compound, drug, or treatment. Using the correlationsmeasured by the mathematical model, gelsolin ELISA using gelsolinbinding agents of the invention is useful to determine clinical outcomeof patients.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and compositions within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

Other embodiments are set forth within the following claims.

1. A method for determining the presence of, or predisposition to, adisease or condition associated with altered levels of gelsolin in amammalian subject, the method comprising the steps of: (a) contacting aurine sample from the mammalian subject under immunologically-reactiveconditions with one or more antibodies or antibody-related polypeptideshaving the same antigen-binding specificity as antibodies produced by adeposited cell line selected from the group consisting of: CGMCCAccession Nos: 2114, 2116, 2247 and 2248; and (b) detecting the bindingof the one or more antibodies or antibody-related polypeptides to thegelsolin-like polypeptide to determine the level of gelsolin-likepolypeptide in the sample, wherein a difference between the level ofgelsolin-like polypeptide in the sample and a reference level indicatesa presence of, or predisposition to, a disease or condition associatedwith altered levels of gelsolin in the mammalian subject.
 2. The methodof claim 1, wherein the sample is contacted with the antibodies orantibody-related polypeptides in an ELISA.
 3. The method of claim 2,wherein the step of contacting comprises binding a first antibody to asubstrate and contacting the sample and a second antibody to thesubstrate, wherein the second antibody comprises a detectable label. 4.The method of claim 3, wherein the first antibody binds to the sameantigenic determinant as an antibody produced by a hybridoma cell lineCGMCC Accession No: 2247 and the second antibody binds to the sameantigenic determinant as an antibody produced by a hybridoma cell lineselected from the group consisting of CGMCC Accession No.
 2116. 5. Themethod of claim 1, wherein the disease or condition associated withaltered levels of gelsolin is selected from the group consisting of:kidney failure, septic shock, multiple organ dysfunction syndrome,rheumatoid arthritis, stroke, heart infarction, cancer, systemicautoimmune disease, chronic hepatitis, side-effects of chemotherapy, andside-effects of radiation therapy.
 6. The method of claim 1, wherein thegelsolin-like polypeptide is a C-terminal fragment of gelsolin.
 7. Themethod of claim 2, wherein an increase in the level of gelsolin-likepolypeptide in the sample compared to the reference level indicates apresence of, or predisposition to, a disease or condition associatedwith altered levels of gelsolin in a mammalian subject.
 8. The method ofclaim 1, wherein the reference level is the amount of the gelsolin-likepolypeptide in a control population of subjects that do not have adisease or condition associated with altered levels of gelsolin.
 9. Themethod of claim 1, wherein the reference level is the amount of thegelsolin-like polypeptide in a subject that does not have a disease orcondition associated with altered levels of gelsolin.
 10. The method ofclaim 1, wherein the reference level is the amount of the gelsolin-likepolypeptide in the subject at an earlier time.
 11. The method of claim 1further comprising quantifying the amount of the gelsolin-likepolypeptide in the sample from the subject.
 12. The method of claim 11further comprising correlating the amount of gelsolin-like polypeptidein the sample from the mammalian subject to a likelihood of survival ofthe subject.
 13. The method of claim 3 further comprising the step of:comparing the level of gelsolin-like polypeptide in the mammaliansubject to a reference level, wherein the reference level comprises acontrol subject not having kidney failure, and wherein an increase inthe level of gelsolin-like polypeptide in the subject compared to thereference level indicates that the mammalian subject has kidney failure.14. The method of claim 3 further comprising the steps of: assigning thesubject to a subject class based on the level of gelsolin polypeptide ofthe subject; and selecting a prophylactic or therapeutic treatment basedon the subject class.
 15. An antibody or antigen-binding fragmentthereof having the same antigen-binding specificity of antibodiesproduced by a deposited cell line selected from the group consisting of:CGMCC Accession Nos: 2247 and
 2248. 16. The antibody or anantigen-binding fragment of claim 15 comprising at least heavy chainCDR3 amino acid sequence selected from the group consisting of:GASEAEKTGA (SEQ ID NO.: 5), or a variant thereof having one or moreconservative amino acid substitutions. 17-24. (canceled)